Apr 13, 2026
Bisulfite-seq for Astrocytes
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Bisulfite-seq for Astrocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld4ex8l5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 13, 2026
Last Modified: April 13, 2026
Protocol Integer ID: 314959
Keywords: ASAPCRN, seq for astrocytes bisulfite, astrocytes bisulfite, astrocyte, bisulfite, seq
Abstract
Bisulfite-seq for Astrocytes
Cell Collection & DNA Extraction
Harvest astrocytes using 1 mL TrypLE Express per well of a 6-well plate
Centrifuge cells and process for column-based DNA purification
Extract genomic DNA using the QIAamp DNA Mini Kit (Qiagen) following manufacturer's instructions
Elute DNA in 80 µL; quantify prior to submission (expected range: 51–198 ng/µL)
Sequencing Submission
Submit purified DNA to Zymo Research for Reduced Representation Bisulfite Sequencing (RRBS)
Library Preparation & Sequencing (performed by Zymo Research)
Reduced Representation Bisulfite Sequencing (RRBS)
Bisulfite conversion efficiency exceeded 99% in all samples
Data Processing
Adapter trimming and quality control with CutAdapt (RRID:SCR_011841)
Alignment to reference genome hg38 using Bismark (RRID:SCR_005604)
Methylation analysis in R using methylKit
Differential methylation assessed using logistic regression with FDR correction
Differentially methylated regions annotated using UCSC genome annotations