RNA library preparation part 2

Continuing RNA extractions

A quick recap of things I’ve revised

RNA library preparation part 1

Analyzing Catlin’s data

Revising the water temperature analysis

RNA extractions

Population comparison for MA and WA crabs

Revising modeling for 2024 and Julia experiments

Water temperatures for WA and Julia’s experiments

Continuing with Catlin’s crabs

Making figures for the manuscript

Revising WA TTR data

Samples for metabolomics and lipidomics

Finishing WA crabs and starting on Catlin’s crabs

Re-genotyping MA crabs and finishing WA crabs

Revising figures and numbers in text

Re-genotyping MA crabs

Temperature conditions during the MA experiment

Hardy-Weinberg equilibrium for MA and WA populations

Re-analyzing Julia’s data

Preliminary WA TTR analysis with genotype

Revising the MA TTR analysis

Interpreting metabolomic and lipidomic WCNA correlations

WA genotyping + some MA troubleshooting (because of course)

Supplementing the metabolomics analysis

Finishing MA genotyping

Returning to ACTUAL genotyping

Supplementing the lipidomics analysis

Returning to genotyping troubleshooting (Part 2)

Returning to genotyping troubleshooting

Revising figures

Correlating WCNA modules

Integrating all analyses

Digging into enrichment data

Linear mixed effects models for TTR

Pairwise metabolomics enrichment

Pairwise lipidomics enrichment

Lipidomics enrichment

Lipidomics WCNA

Fixing DMR figures

Reworking lipidomics analysis

(Somewhat) finalized metabolomics figures

(Somewhat) finalized pathway analysis methods

Refining a priori metabolomics analysis

DMR figures

Analyzing WA crab TTR data

BAT_correlating

DMR-DEG overlaps

Trying some lipid blanks standards

Experiment log: WA experiment

Developing a lipid assay

Experiment log: WA acclimation

Analyzing TTR data for MA crabs

Genotyping MA crabs (back in troubleshooting land)

Genotyping MA crabs (but now it’s hell)

Experiment log: WA crabs!

Experiment log: experiment week!

Genotyping MA crabs

Experiment log: Acclimation week

Characterizing DMR locations

General methylation landscape figure

We have experimental animals!

Testing the restriction digest

Temperature tweaks and setting up the cold room

PCR optimization

Picking a restriction enzyme

Making it even colder

Testing new primers

Finishing genotyping

Checking on temperatures

Designing a restriction digest

Characterizing the general methylation landscape with the new genome

Returning to the killifish project (again)

Starting the 2024 experiments

Troubleshooting sample 72

Rerunning samples on a gel

Extraction, PCR, and gel for remaining crabs

Extraction, PCR, and gel for remaining crabs

Extraction, PCR, and gel for remaining crabs

Extraction, PCR, and gel to “redo” crabs

Investigating specific metabolite and lipid pathways

Continued layover in contamination station

Results from genotyping round 2

Contamination station round 3

PCR and gel for TTR crabs

Extractions, PCR, and gel for TTR crabs

Extractions, PCR, and gel for TTR crabs

Some notes

Enrichment, enrichment

Analyzing changes in the maximum number of transcripts expressed

Counting DML in genes with a predominant isoform shift

Extractions, PCR, and gel for TTR crabs

Extractions, PCR, and gel for TTR crabs

Continuing gene activity analyses

Extractions, PCR, and gel for TTR crabs

Extractions, PCR, and gel for TTR crabs

PCR and gel for TTR crabs

Predominant isoform shift model

Extractions, PCR, and gel for respirometry + TTR crabs

Extractions, PCR, and gel for respirometry + TTR crabs

Testing out new SMC primers

Testing out new SMC primers

GLM for respirometry data

Individual-level analyses

Experimental design edits

A reason for decontamination station

Simplifying the demographic data analysis

Still at decontamination station

Moving past decontamination station (for now)

Revising analyses

Preliminary lipidomics analysis

Functional metabolomics analysis

Multivariate metabolomics analysis

Revisiting genotypes

Decontamination station (my least favorite stop on the extraction train)

Making a plan for metabolomics analysis

Extractions, PCR, and gel for respirometry + TTR crabs

Modifying respirometry analysis

Incorporating genotype into performance assays

Genotyping respirometry crabs

Preliminary analyses for 2023 experiment

Re-doing extractions, PCR, and gel for respirometry crabs

Extractions, PCR, and gel with third set of respirometry crabs

Extractions, PCR, and gel with second set of respirometry crabs

Re-trying extractions, PCR, and gel

Revising respirometry analysis

Re-trying PCR

Starting chelex extractions for genotyping

Analyzing respirometry data (finally)

Treatment Days 44-49

Treatment Days 40-43

Treatment Days 33-39

Treatment Days 27-31

Treatment Days 23-26

Treatment Days 21-22

Treatment Days 16-20

Treatment Days 14-15

Treatment Days 10-13

Treatment Days 7-9

Treatment Days 1-6

Acclimation Day 4 and 5

Experiment is UNDERWAY

The final countdown (for the Hawaii paper)

Finalized facility set-up

Starting facility set-up

Finishing the demographic data analysis

Experiment planning update!

Waterproof labelling system (I hope)

Polynomial terms in transcriptional noise analysis

Time-to-right analysis

Survival analysis

Transcriptional noise analysis

Analyzing demographic data

Pathway analysis for RNA-Seq data

Global methylation differences by population

Alternative splicing analyses

Gene methylation and expression mixOmics

Correlating gene methylation and expression

Continuing with the new genome

Re-align to the new genome

Temperature conditions within tanks

Treatment Days 18 through 22

Treatment Days 15 through 17

Treatment Days 11 through 14

Genomic locations of DML

Treatment Days 8 through 10

Treatment Days 5 through 7

Treatment Days 3 and 4

Treatment Days 1 and 2

Acclimation Day 5 and 6

Acclimation Day 3 and 4

Acclimation Day 2

Final set up + Day 1!

Setting up the experimental facility

Wrapping up the initial methylation analysis

Digging into DMR

Respirometry trial

Removing L4 samples

Methylation landscape information

Revised DMR identification

Gene expression variability analyses

Revisiting DMR identification

Messing around with DMR identification

Methylation landscape analysis

Identifying DMR

Figuring out my BAT_overview issues

Moving forward with summarizing methylation

General methylation landscape comparisons

Methylation extraction results

Extracting methylation information

Trying to tune the sPLS

Continuing sample alignment

Full sample alignment

Exon methylation and expression

Continuing mapping tests

Mapping RRBS data

Documenting for posterity (because it’s an annoying process)

Thinking about SNPs

Trimming RRBS data

Starting a new project!

Miscellaneous enrichment investigations

Gene librarian for genes with DML associated with enriched GOterms

Digging into enrichment results

Protocol for care during non-experimental time

Addressing reading committee edits

Miscellaneous analyses

Revising methylKit parameters

Finishing up analyses and figures!

Trying topGO for GOterm enrichment

Figures and statistical analyses

Restarting functional annotation with blastx

Functional annotation for new C. gigas genome

Trying DSS for DML identification

Reworking pH-DML

Running randomization tests on mox

Investigating methylation data

Messing around with methylKit DML settings

Working with BS-Snper SNPs

Characterizing DML

Characterizing general methylation landscape

Comparative analysis with methylKit

Generating genome feature tracks

Troubleshooting R Studio server session timeouts

methylKit with R Studio server on mox

Running R scripts on mox (for real this time)

Running R scripts on mox

Preparing to R scripts on mox

Separating female and indeterminate samples

Testing base methylKit code

Identifying SNPs with BS-Snper

Identifying SNPs

Merging BAM files

Reviewing revised alignments

Processing Roslin alignments

Testing methylKit parameters

Preliminary DML identification

WGS resequencing quote

Preliminary methylation investigation

Reviewing bismark output

Aligning to the new C. gigas genome

Evaluating bismark output

Starting bismark with Manchester data

Sending samples for sequencing

Reviewing trimgalore output with multiqc

trimgalore output and fastqc

Analyzing the full dataset

Reviewing second round of trimming

Revisiting trimming

Checking trimmed data and starting bismark

Trimming Hawaii data

Investigating methylation island characteristics

Final sperm sample extractions

Re-extracting low-yield sperm samples

Extracting DNA and RNA from sperm samples

Testing protocol for sperm samples

Returning to contingency tests

Re-extracting female samples 16 and 35

General methylation calculations

Checking female DNA quality

Checking female RNA quality

Checking female DNA and RNA quality

October Goals Recap

Remaining female DNA and RNA extractions

Subsampling remaining female tissue

Tweaking Zymo MicroPrep Kit protocols

Yields from Zymo MicroPrep Kit test samples

Testing the Zymo MicroPrep Kit (again)

Revisiting the manuscript

Finishing ethanol precipitations

DNA elution and ethanol precipitation (again)

MBD-Biotin wash and binding (again) and ethanol precipitations

DNA elution and ethanol precipitation

MBD-Biotin wash and binding

Final quality check

Final shearing and quality checks

Ethanol precipitations

Checking samples on the BioAnalyzer

Continuing shearing and quality checks

More planning for WGBS

DMC locations and cleaning up scripts

Planning for WGBS

Re-shearing DNA and checking quality

Small tasks to complete the manuscript

Another QC check before enrichment

Additional sample preparation

Prepping for C. virginica sperm MBD Enrichment

Plan of attack for the mountain of labwork

Another (and hopefully final) iteration of union bedgraph analyses

Calculating median percent methylation

Checking stacked barplots and post-hoc tests

Refining multivariate and modeling approaches

Preliminary follow-up GLMs

Multivariate analysis for compositional data

Formatting individual sample information

Fixing characterization code and remaking barplots

Retooling labwork plans

Genome features and running the CpG characterization pipeline (again)

Statistical comparisons for methods

Reworking flanking and intergenic regions

Revamping distribution plots and creating genome feature plots

Characterizing union bedgraphs

More feature tracks

Revisiting CpG distributions

CpG distributions

Characterizing CpGs in 5x data

Small tasks for future analyses

Retroactively obtaining checksums

Characterizing full samples

Modifying the CpG characterization pipeline

Finalizing genome feature tracks

More genome feature tracks

Comparing methylation analysis methods for two coral species

Epigenetics reading group feedback!

Addressing reviewer comments

Reframing my questions and making a poster

C. gigas vs. C. virginica: DML

C. gigas vs. C. virginica: general methylation landscapes

Comparing Crassostrea spp. methylation patterns

C. gigas RNA and DNA extractions: Day 1

Looking at labwork and sequencing options

Gearing up for C. gigas labwork

Tying up loose ends for the C. virginica gonad paper

My last (I promise) analyses

Revising manuscript figures

Adding GOslim annotations to genes

Addressing collaborator feedback

One last set of analyses before dedicated writing

Further DML annotations

Preliminary data for PCSGA

Finalizing the analysis pipeline

Looking at preliminary DML

Rerunning bismark and starting with methylKit

Fixing my bismark script for Mox

Testing bismark parameters for WGBS data

Characterizing GOslim terms

Enrichment and description of differentially methylated genes

Identifying differentially methylated genes

Starting the differentially methylated gene analysis

Revised gene description using GO-MWU information

Gene enrichment with GO-MWU

Things I did at FHL that were not my research

Gene enrichment and description

Gene annotation tables

Reworking DMR

Finalizing C. virginica tracks and revisiting analyses

My recollections of the Functional Re-annotation of Oyster Genomes with Epigenetic Resources (FROGER)

Even more troubleshooting with IGV

Continued troubleshooting

Teaching students about oysters and ocean acidification

Revised finalized general methylation trends

Finalized general methylation trends

Refining general methylation trends

Describing general methylation trends

Revisiting DML analyses

Resolving DML and DMR visualization issues

Visualizing DML and DMR

Requantifying C. virginica sperm DNA extractions

Finishing C. virginica sperm DNA extractions

Continuing sperm DNA extractions

DML-mRNA gene product information

Some information I’ve missed

Isolating and quantifying C. virginica sperm DNA

Extracting DNA from C. virginica sperm samples

DMR-mRNA gene product information

Gene enrichment for mRNA overlaps

Continuing isopropanol precipitations

Preparing C. gigas DNA for WGBS

Examining sample clustering

Proportion test results

Overlaps with methylKit gene background

Using simper and new figures

Final unconstrained and constrained ordinations

Protein abundance and environmental data NMDS

Flanking analysis for mRNA coding regions

Revisiting NMDS for protein abundance data

methylKit and bedtools with Mox samples

Mox update

Preliminary bedtools analysis

Tiling window analysis in methylKit

Different mincov values in methylKit

Running bismark on Mox

Testing methylKit parameters

Sample extraction: Day 2

Testing bismark parameters

Sample extraction: Day 1

Extraction plan for actual samples

Gene Enrichment Results from compGO

DML-mRNA Gene Enrichment Results

DML-mRNA Gene Enrichment

Blasting for Uniprot codes (again)

Protocol test: Round 4

T-24 days until classes…

Plan for another protocol test

Microscopes and the Fish Collection

Teaching students about cryptobenthic reef fish!

Protocol test: Round 3

Protocol test: Round 2

Testing the extraction protocol

Today’s hilarious labwork fail

The last month of this academic year :0

Assumptions and p-values

lmers and glmers

Blasting for Uniprot codes

flankBed 101

Quantifying proportion of overlaps

Understanding gene enrichment

Finding overlaps between DMLs and other things

Another month, another set of dreams

Revised salinity data!

Preparing for discoveries

Note to self: Always double check things

A new enchilada

A revised enchilada

Revising environmental data

A spoiled enchilada

The whole enchilada (but better this time)

Reproducibility rodeo

I am Jerry Gergich

Visualizing bismark outputs

The whole enchilada

What are those?!

The meme has evolved

CCC: Carbonate Chemistry Calculations

M&M: methylKit and MACAU

Having pH in mV makes no sense

The end of a pipeline test

When the stars (and sequences) align

Preparing a genome (and my brain)

Quality: controlled

Yaamini vs. Computers

Time to analyze the C. virginica data

I have water chemistry data!

Things to do if I had infinite research money

April showers = More writing hours

The curious case of the catalase(s)

Do I have a story for my larval data?

Another classification revision

GLMs are neat

I think I understand classification better now

[Insert something witty about March Madness]

This series has (finally) come to an end

Digital drawing board: Round 2

My better figure wasn’t actually good

I created a better figure!

Tying up (more) loose ends

i.e. My grand return to RStudio

The many paths to school

Taught sixth grade science today!

Tryna make the most out of this short month

Rinse and repeat

Dynabeads are dynamite

A fulfilling day of labwork!

I pimped out my notebook!

All systems go on C. Virginica sequencing!

Histology is not an exact science

I guess break is over…

The growth data wasn’t very interesting

I see the light

Triple-nested for loops are not fun

a.k.a. How to take a break from proteomics

The last bit of data!

Yesterday’s meeting notes

Some more regressions

Connecting the dots

Following through on my plan

Quick journey into Manchester world

My final NMDS

Visualizing environmental and biomarker data

Column comparisons

More data, more problems…?

Some more information from Micah and Alex

Looked into ROC curves

I’ve got the power

Investigating those bad samples

All lab notebook links are now fixed

Science doesn’t take a day off

I have significant results?!

Everything at the peptide level

Squeezing every last bit of information out of my data

The end of a saga

a.k.a. An excuse to make mini apple pie cheesecakes

Better results?

Maybe I should have called this series “Troubleshooting”…

I finally have technical replication I kinda trust?!

Dead ends and new plans

Things DO get better! (Kind of)

Continuing the PRTC confusion

I tried doing something statistically impossible today

Step 1: R-squared Cutoffs

Are some transitions worse than others?

R&R: Review and Reproduce

We interrupt your regularly scheduled lab notebook perusing for some maintenance.

One script, infinite boxplots

Another Skyline saga

Retracing my steps

Everything sucks.

R Help is Helpful

All aboard the NMDS Struggle Bus

…Everybody Everywhere

Clean up, clean up

The end.

We have a Game Plan

The Final Countdown

They’re still not growing!

Checked the 80 micron screen again

Totes are pesky

Immersion heaters are pesky

Anoxia is no bueno

Smooth Saturday screening

Lots of maintenance today

My larvae are 60 microns now!

Counting larvae takes a while

A bit of a rough start today

I only have five goals

I’m the Mother of Oysters

Spoiler: We have baby oysters!

NO OYSTERS DIED!

It’s getting ugly

The mortalities continue

Revised for the last time

My prior calculations were incorrect!

Featuring reduced crosses and pools

Temperature is at 23 ºC!

If you don’t have a visualization, did you really analyze data?

My samples are DONE.

Last day of my samples!

Status quo?

Murphy’s Law is always relevant

The power is back!

Still plugging along

Power outage edition

More borrowed PRTC = All samples prepared

Our assay works!

It’s SRM assay time!

WE HAVE OUR TARGETS.

Those peaks are running interference

And the pH is low (again)

I have draft transitions!

Differential Expression + Protein Function = Targets?

The pH is kind of stable?

MSstats works!

June was rather slow

It’s time to think about crosses!

Revised (final?) Conditioning Plan

Sophie’s New Choice: Temperature, Flow or pH?

It’s been a slow burn

I was gifted 3,000 extra proteins

This time it only took 4 hours!

Remember that Skyline fail?

From Saturday 6/10 to Wednesday 7/5

Last day of DNR extractions (hopefully)!

Time to fatten the oysters up!

It’s getting hot in here

Yaamini vs. CentriVap: The Sequel

(Almost) done with Spring Quarter already?!

I am now a faster pipetter

All this protein but I’m still not swole.

Here’s hoping this is the last round of DNR sample preparation!

I need to become a faster pipetter.

We have lots of protein!

How many times can you restart sample preparation?

Slice and dice

More cowbell? More samples.

The DIA pipeline has been quite a #strugglebus

It’s always good to practice your methods of torture.

Who wants to manually quality check peptides?!

Record scratch. Freeze frame.

Who are the lucky winners?

It’s time to extract more protein.

I can now analyze my full .blib!

It’s already MAY.

Skyline works with our demultiplexed files!

It’s not as easy as clicking a button

Laura went out to Manchester today and graciously cleaned my oysters

I became well-aquainted NCBI SRA database today

I’m going to get stuff done for Pubathon (I promise)!

My files are done converting!

Another new MSConvert CLI

New MSConvert CLI, hot off the presses

Demultiplexing is perplexing

OA System Breakdown

We’re done with our OA experiment!

I was so busy catching up on classes I forgot it was April!

What happened when Laura got stranded in the Midwest

I’m at NSA, to Grace and Olivia went to Manchester.

Here’s the evolution of my poster for NSA!

Checking in before we leave for NSA

On that number-crunching grind

We have data!!!

A couple hiccups at Manchester today

It works!

Yesterday I set up a system for cataloging our water chemistry data.

Reformatting PECAN inputs

New day, same errors

We’re officially halfway through our pH treatment window!

To reconvert or not reconvert?

Hit an obstacle with PECAN this weekend.

Another smooth day at Manchester!

:cake: It’s not as easy as pie :cake:

March Maddness is upon us.

Today, I spent 3 hours cleaning oyster :poop:

The OA System is up and running!

February Goals Update

Conducted my first outreach activity today!

First Manchester Sampling

February Goals

…But now trouble is gone!

…And trouble stuck around at 3:51 p.m. on January 29.

Trouble struck at 8:16 p.m. on January 28.

My samples are on the Mass Spec!

Mass Spectrometer Set-up

M/Z ratios: Mass hysteria ensues

PRTC: We know what those peptides can be

Not Salty about Desalting

Speed Vacuum = Slow Process

Trippin for Trypsin

If at first you don’t succeed, trial trial again.

Practice makes perfect?

Gill Tissue + Sound Waves = Protein Soup

My first day in lab was a busy one!

My DNR project is still on the brain!.

Now that it’s almost November, it’s time to get cracking on the DNR project.

As promised, here is some background on my current project

Hello!