---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-02-28 00:36:58+00:00
layout: post
slug: bs-seq-library-prep-c-gigas-larvae-oa-1000ppm
title: BS-seq Library Prep - C.gigas Larvae OA 1000ppm
categories:
  - 2015
  - Crassostrea gigas larvae OA (2011) bisulfite sequencing
tags:
  - bisulfite conversion
  - BS-seq
  - Crassostrea gigas
  - EpiNext Post-Bisulfite DNA Library Preparation Kit
  - larvae
  - library prep
  - Methylamp DNA Modification
  - NanoDrop1000
  - OA
  - ocean acidification
  - Pacific oyster
---

### Bisulfite Conversion



Pooled 200ng each of the sheared 1B1 (4μL) & 1B2 (used the entire sample, 20μL) 5.13.11 1000ppm _C.gigas_ larvae DNA samples for a total of 400ng. Total volume = 24μL.

Quantified the pooled DNA using the NanoDrop1000 (ThermoFisher) prior to initiating bisulfite conversion.

![](https://eagle.fish.washington.edu/Arabidopsis/20150227_Emma_1000ppm_pool_preBS_plot.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150227_Emma_1000ppm_pool_preBS_plot.JPG)

Clearly, the NanoDrop measurements differ from the expected concentration. NanoDrop suggests the total amount of input DNA is ~1400ng (58ng/μL x 24μL = 1392ng). This is most likely due to RNA carryover, as [DNA quantification using a fluorescence-based, double-stranded DNA assay performed previously shows a drastically lower concentration](https://robertslab.github.io/sams-notebook/posts/2015/2015-01-13-speedvac-c-gigas-larvae-oa-dna/).

Proceeded with bisulfite conversion using the Methylamp DNA Modification Kit (Epigentek) in 1.5mL tube, according to the manufacturer's protocol:




    
  * Added 1μL to sample, incubated 10mins @ 37C in water bath

    
  * Made fresh R1/R2/R3 solution (1.1mL R3 buffer added to vial of R2, vortexed 2mins, 40μL R1 added to mixture - Remainder stored @ -20C in "-20C Kit Components Box")

    
  * Added 125μL of R1/R2/R3 solution to sample, incubated 90mins @ 65C in heating block with water

    
  * Addd 300μL R4 to sample, mixed, transferred to column, spun 12,000RPM 30s

    
  * Added 200μL R5 to column, spun 12,000RPM 30s

    
  * Added 50μL R1/ethanol solution to column, incubated 8mins @ RT, spun 12,000RPM 30s

    
  * Washed column with 200μL of 90% EtOH, spun 12,000RPM 30s; repeated one time.

    
  * Eluted DNA with 12μL R6, spun 12,000RPM 30s



Quantified post-bisulfite-treated sample on NanoDrop1000:

![](https://eagle.fish.washington.edu/Arabidopsis/20150227_Emma_1000ppm_pool_postBS_plot.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150227_Emma_1000ppm_pool_postBS_plot.JPG)

Definitely a low yield (~108ng) relative to the input (~400ng). Will proceed with Illumina library prep.





### Library Prep



Illumina library prep was performed with [EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek)(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/Epigentek_PostBisulfiteIlluminaLibraryPrep_P-1055.pdf).  Changes to the manufacturer’s protocol:




    
  * Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.

    
  * PCR cycles: 15



No other changes were made to the manufacturer’s protocol.

Epigentek Barcode Indices assigned, per their recommendations for using two libraries for multiplexing (this will be combined with the 400ppm library):

Barcode #12 – CTTGTA

The library was stored @ -20C and will be checked via Bioanalyzer on Monday.