ChIP-Atlas: SRX8524462

Antigen

Cell type Class: Neural
Cell type: Astrocytes
MeSH Description: A class of large neuroglial (macroglial) cells in the central nervous system - the largest and most numerous neuroglial cells in the brain and spinal cord. Astrocytes (from "star" cells) are irregularly shaped with many long processes, including those with "end feet" which form the glial (limiting) membrane and directly and indirectly contribute to the BLOOD-BRAIN BARRIER. They regulate the extracellular ionic and chemical environment, and "reactive astrocytes" (along with MICROGLIA) respond to injury.

library_strategy: ATAC-seq
library_source: GENOMIC
library_selection: other
library_construction_protocol: ATAC sequencing libraries were prepared using the OMNI-ATAC protocol (Corces et al., 2017) with minor modifications. 25,000 MACS-purified astrocytes or dissociated cultured cells were collected by centrifugation (300g, 5 min, 4C), and permeabilized for 3 min on ice in 50 ul ATAC-RSB buffer (Tris/HCl (pH7.4) 10 mM, NaCl 10 mM, MgCl2 3 mM) containing 0.1 % NP-40 (IGEPAL CA-630), 0.1% Tween-20 and 0.01% digitonin. 1 ml ATAC-RSB buffer containing 0.1 % Tween-20 was added and the nuclei were recovered by centrifugation (500g, 10 min, 4C). The pellet was resuspended in 25 ul of transposase reaction mix (12.5 ul 2x TD buffer and 1.25 ul Tn5 transposase (Illumina Cat #FC-121-1030), 8.25 ul PBS, 2.5 ul water, 0.1% Tween-20, 0.01% digitonin), and incubated on a rotational shaker (1000 rpm) for 30 min at 37C to fragment and tag accessible chromatin. DNA was then purified using the “MinElute PCR purification kit” (Qiagen, Cat no. 28004) and eluted in 20 ul EB buffer (Qiagen). 5 ul DNA were were amplified by PCR in 20 ul reactions using NEBNext HiFi 2x PCR Master Mix (New England Biolabs, M0541) and barcoded primers described previously (Buenrostro et al., 2013) for 12 cycles: (1) 5 min 72C, (2) 30s 98C, (3) repeat 12x: 10s 98C, 30s 63C, 60s 72C. The amplified libraries were purified using SPRIselect beads (ratio sample : beads 1 : 1.8)