title: GSM7669582: Subject_4 Oligodendrocyte Precursor Cells H3K27ac ChIP-seq Input; Homo sapiens; ChIP-Seq
library_name: GSM7669582
library_construction_protocol: A novel method to isolate OPC and MO nuclei by FANS from frozen postmortem brain tissue was used. The protocol includes the following steps: (1) tissue specimens are homogenized at 4C in 4 ml of lysis buffer with a glass Douncer, (2) the homogenates are underlaid with a 4.5 ml layer of high sucrose buffer, and centrifuged at 24,000 rpm for 1 hour, (3) nuclear pellets are resuspended in 300 ul of blocking buffer and incubated with primary antibodies for 1.5 hours while rotating at 4C, (4) another centrifugation step for 30 min through a layer of high sucrose buffer is performed, (5) nuclear pellets are resuspended in blocking buffer and incubated with secondary antibodies for 1 hour, (6) after diluting the samples 5x times, a DNA dye (DAPI) is added, and the samples are separated by FANS on a flow cytometer. The following reagents, parameters and modifications to the basic protocol were employed. Primary antibodies were against the targets: NeuN (also known as RBFOX3) – a well-established marker of neuronal nuclei, SOX10 – a marker of oligodendroglial lineage, and SOX6 – a marker of OPCs. For anti-SOX10 antibodies (goat polyclonal, R&D Systems, AF2864), custom-made direct conjugates with the AF647 fluorophore were prepared and used at 1:150 dilution. For anti-NeuN antibodies, commercially available direct conjugates with the phycoerythrin (PE) fluorophore were used (mouse monoclonal anti-NeuN-PE, Millipore, FCMAB317PE, at 1:1000 dilution). For anti-SOX6 antibodies [guinea pig polyclonal antibodies, 1:1000 dilution (Stolt et al., 2006)], anti-guinea pig AF488-conjugated secondary antibodies (Jackson ImmunoResearch, 706-546-148) were used at a 1:250 dilution during the second incubation step. The use of the flow cytometer instrument BD Influx with four lasers (355nm, 488nm, 561nm and 640nm) allowed for separation between sorted populations with no need to perform compensation. The details of the gating steps in the flow cytometry protocol are provided in the Results section (see also Fig. 1). In the experiments aimed at obtaining nuclei for ChIP, protease inhibitors (benzamidine (Sigma) and PMSF (Thermofisher Scientific) at 1:1000 dilutions from 0.1 M stocks) were added to the lysis buffer. We employed the native ChIP protocol (N-ChIP) in which chromatin fragmentation is performed using micrococcal nuclease (MNase) without crosslinking proteins to DNA (Sci Adv. 2018). We used anti-H3K27ac antibodies from Active Motif (Cat# 39133; rabbit polyclonal, 3 µg per sample). ChIP-Seq libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Cat. # E7645). The resulting libraries were sequenced on a NovaSeq 6000 instrument (Illumina), using paired-end 100 cycles protocol.