---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: Library Decisions - C.bairdi RNAs for Library Pools
date: '2019-05-14 14:38'
tags:
  - tanner crab
  - RNA
  - Chionoecetes bairdi
categories:
  - 2019
  - Tanner Crab RNAseq
---
I isolated a bunch of tanner crab RNA on [20190430](https://robertslab.github.io/sams-notebook/posts/2019/2019-04-30-RNA-Isolation-and-Quantification---C.bairdi-Hemolymph-Pellet-in-RNAlater/) and Steven asked me to try to figure out some options for RNAseq libray pools.

Using Grace's table join of my Qubit data ([hemo_qub-for-libs.csv](https://raw.githubusercontent.com/RobertsLab/project-crab/8f95500019075d917e9cee013af325cf3a7d0bda/analyses/hemo_qub-for-libs.csv)), I created a pivot table to quickly get an idea of how things looked (note: I added a column with total RNA yield for each sample - 10uL * concentration):

![C.bairdi pivot table screen cap](https://github.com/RobertsLab/sams-notebook/blob/master/images/screencaps/20190514_cbai_library_table.png?raw=true)


Assuming a minimum of 1000ng is required by the [UW's Northwest Genomics Center ](https://nwgc.gs.washington.edu/) (that was the requirement last time we had RNAseq performed by them; have emailed to confirm it's still required), here are the libraries I think we can/should make.

Possible libraries, by "Day":

- Day 9

  - Infected vs Uninfected

- Day 12

  - Infected vs Uninfected
  - Ambient vs Cold vs Warm

- Day 26

  - Infected vs Uninfected
  - Ambient vs Cold


The "Day 9" samples are named poorly, as these are actually the samples prior to temperature exposures. Thus, the "trtmnt_tank" column (i.e. temperature treatment) in the table above doesn't really apply. With that in mind, Day 9 samples can only be compared as Infected/Uninfected.

So, by default, those should be two libraries.

Then, we have the Day 12 and Day 26 samples. We can either do Infected/Uninfected _OR_ Ambient vs. Cold, but can't do both, as there isn't sufficient RNA...

Admittedly, I'm not sure which aspect of this project was given more weight in the grant proposal:

- response to disease over time
- response to temperature over time

The answer to that question will guide library pooling.

With that said, if we do the following, we'll have a bit of both pathogen response and temperature response:

- Day 9 Infected/Uninfected

- Day 12 Ambient/Cold

- Day 26 Ambient/Cold
