| GSM7669618 channel 1 extraction |
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 OPC and MO nuclei for RNA isolation, RNase inhibitors were added to all buffers during the nuclei preparation protocol (Recombinant RNase Inhibitors, Takara; 1:40 dilution). For RNA-seq library construction, Pico Input Mammalian SMARTer® Stranded Total RNA-Seq Kit v2 (Takara) was used, with 10 ng RNA as input material and 12 cycles of PCR amplification. The SMARTer® Total RNA-Seq protocol includes a ribosomal cDNA depletion step. Libraries were sequenced on a NovaSeq S4 instrument (Illumina) using the paired-end 100-cycle (PE100) protocol to the average depth of ~50 million reads per sample. |
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