---
title: "Tissue subsampling for WGBS (methylation work)"
author: "Kathleen Durkin"
date: "2024-04-25"
categories: ["pacifc-cod"]
format:
  html
---

We're planning on sending out samples for whole genome bisulfite sequencing (WGBS) to examine methlation in our cod samples. Today I subsampled all of the liver tissue.

[Sample list](https://docs.google.com/spreadsheets/d/19CqqS3wOhmG7drw163v7evUylRWwOjBOdvuR-y0RqF0/edit#gid=155247170) (liver samples are firm, but we're still choosing blood and gill samples)

### Sample selection

All samples selected for methylation work are a subset of samples that were RNA sequenced. In sample selection we took consideration of RNA extraction and sequence quality, tank, genetic variability, size, and ZP3 gene haplotype.

40 total samples

-   32 liver (8/treatment)

-   4 blood (2 from 0\*C, 2 from 16\*C)

-   4 gill (2 from 0\*C, 2 from 16\*C)

### Subsampling protocol

Sterilized all tools in 10% bleach, then rinsed in DI water before use. Sterilized between every use.

Tissue quantity recommended by Azenta for DNA extraction from whole tissue is 10-30mg. Since all of these samples have already been subsampled for RNAseq work, some of the samples had little tissue left. Any sample with \>15mg of tissue was subsampled, selecting at least 10mg for methylation work. For any sample with \<15mg of tissue left, the entire remaining tissue was sampled for WGBS. For 15 of the liver samples, no tissue remained after subsampling for WGBS.

After subsampling, methylation liver samples were stored in a box labelled "P.cod samples for methylation" with the date (04/25/2024) and my initials (KMD) in the middle -80 freezer in FTR.

### WGBS Samples

|                  |                  |                  |                   |
|------------------|------------------|------------------|-------------------|
| **0C treatment** | **5C treatment** | **9C treatment** | **16C treatment** |
| 39               | 118              | 79               | **3**             |
| **40**           | **119**          | **80**           | **4**             |
| **47**           | 120              | 88               | **11**            |
| 50               | 127              | **94**           | 12                |
| 57               | 131              | 97               | **18**            |
| 59               | **138**          | **99**           | **20**            |
| 69               | 148              | 108              | 29                |
| **70**           | **156**          | **116**          | 30                |

Samples that had no remaining tissue after WGBS subsampling (bolded above): 3, 4, 11, 18, 20, 40, 47, 70, 80, 94, 99, 116, 119, 138, 156

![All methylation liver subsamples](images/IMG_8631.jpg)

![Samples with no remaining tissue post-subsampling](images/IMG_8635.jpg)
