Apr 14, 2026
ATAC-seq Protocol for Astrocytes
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. ATAC-seq Protocol for Astrocytes . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrbzn3lmk/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 14, 2026
Last Modified: April 14, 2026
Protocol Integer ID: 314958
Keywords: ASAPCRN, seq protocol for astrocyte, astrocyte, seq protocol, atac, seq
Abstract
ATAC-seq Protocol for Astrocytes
Cell Collection
Cell Collection
Remove media from 2D astrocyte cultures
Scrape cells in ice-cold PBS
Centrifuge at 1,000 × g for 5 minutes, all steps kept on ice
Ship pelleted cells to Azenta/GENEWIZ for library preparation and sequencing
Library Preparation
Library Preparation (performed by GENEWIZ)
Live cell samples thawed, washed, and treated with DNase I (Life Tech, Cat. #EN0521) to remove genomic DNA contamination
Cell viability and quantification assessed using a Countess Automated Cell Counter (ThermoFisher Scientific)
Nuclei isolated by cell lysis and cytosol removal
Nuclei tagmented with Tn5 enzyme (Illumina, Cat. #20034197) for 30 min at 37°C
Tagmented DNA purified with Minelute PCR Purification Kit (Qiagen, Cat. #28004)
Libraries barcoded with Nextera Index Kit v2 (Illumina, Cat. #FC-131-2001) and amplified by PCR, followed by SPRI bead cleanup
Sequencing
Sequencing (performed by GENEWIZ)
Illumina HiSeq 4000 (or equivalent)
Paired-end, 2x150 bp reads
Raw .bcl files converted to FASTQ and demultiplexed using bcl2fastq 2.17 (1 mismatch allowed)
Data Processing
Data Processing (performed by GENEWIZ)
Quality control and adapter/low-quality base trimming with Trimmomatic 0.38
Alignment to reference genome GRCh38 using bowtie2
Filtering with samtools 1.9: minimum mapping quality ≥ 30, concordantly aligned, primary alignments only
PCR/optical duplicate removal with Picard 2.18.26
Mitochondrial reads and unplaced contig reads removed prior to peak calling
Peak calling with MACS2 2.1.2 to identify open chromatin regions
Peaks present in ≥ 66% of samples per group retained for downstream analysis
Pairwise comparisons: peaks from both conditions merged; reads under peaks counted
Differential peak analysis using DiffBind (R)