alias: GSM4271860
title: GSM4271860: S1_MGAS_ACC; Homo sapiens; ATAC-seq
library_construction_protocol: 50mg of frozen brain tissue was homogenized in cold lysis buffer (0.32M Sucrose, 5 mM CaCl2, 3 mM Magnesium acetate, 0.1 mM, EDTA, 10mM Tris-HCl, pH8, 1 mM DTT, 0.1% Triton X-100) and filtered through a 40µm cell strainer. The flow-through was underlaid with sucrose solution (1.8 M Sucrose, 3 mM Magnesium acetate, 1 mM DTT, 10 mM Tris-HCl, pH8) and subjected to ultracentrifugation at 24,000 rpm for 1 hour at 4°C. Pellets were resuspended in 500µl DPBS and incubated in BSA (final concentration 0.1%) and anti-NeuN antibody (1:1000, Alexa488 conjugated, Millipore Cat #MAB377X), anti-SOX67 and anti-SOX108 as previously described4 Prior to FANS sorting, DAPI (Thermoscientific) was added to a final concentration of 1µg/ml. GABAergic neurons (DAPI+ NeuN+ SOX6+), Glutamatergic neurons (DAPI+ NeuN+ SOX6-), oligodendrocytes (DAPI+ NeuN- SOX10+) and microglia/astrocytes (DAPI+ NeuN- SOX10-) nuclei were sorted into individual tubes (pre-coated with 5% BSA) using a FACSAria flow cytometer (BD Biosciences). ATAC-seq reactions were performed on the 48 samples using an established protocol51 with minor modifications. In brief, 75,000 sorted nuclei were centrifuged at 500 g  for 10 min at 4°C. Pellets were re-suspended in transposase reaction mix (25 μL 2x TD Buffer (Illumina Cat #FC-121-1030) 2.5 μL Tn5 Transposase (Illumina Cat #FC-121-1030) and 22.5 μL Nuclease Free H2O) on ice. Reactions were incubated at 37°C for 30 min and then purified using the MinElute Reaction Cleanup kit (Qiagen Cat #28204), eluting in 10µL of buffer EB. Following purification, library fragments were amplified using the Nextera index kit (Illumina Cat #FC-121-1011) under the following cycling conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermocycling at 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute for a total of 5 cycles. To prevent saturation due to over-amplification, a 5 µl aliquot was then removed and subjected to qPCR for 20 cycles to calculate the optimal number of cycles needed for the remaining 45 µL reaction. The additional number of cycles was determined by first plotting linear Rn vs. Cycle  and secondly calculating the cycle number corresponding to 1⁄4 of maximum fluorescence intensity. In general, adding 2-4 cycles to this estimate was found to yield optimal libraries, as evidenced by analysis on Tapestation D5000 ScreenTapes (Agilent technologies Cat# 5067-5588). Following amplification, libraries were resolved on 2% agarose gels and fragments ranging in size from 100-1000bp were excised and purified (Qiagen Minelute Gel Extraction Kit – Qiagen Cat#28604). Prior to sequencing, libraries were quantified with the Qubit dsDNA HS assay kit (Invitrogen Cat#Q32851) and by quantitative PCR (KAPA Biosystems Cat#KK4873). Fragment sizes were estimated using Tapestation D5000 ScreenTapes (Agilent technologies Cat# 5067-5588) and libraries were sequenced on Hi-Seq2500 (Illumina) obtaining 2x50 paired-end reads.
identifiers.namespace: GEO
identifiers.id: GSM4271860