GSM8729911: NBB_C11_AG; Homo sapiens; RNA-Seq
FACS sorting of PU.1+ nuclei from postmortem samples. Single-nuclei RNA-Seq of FACSsorted nuclei suspensions was performed on Chromium instrument (10X genomics) following the user guide manual (Reagent Kit 3' v3.1). Each sample, containing approximately 10,000 nuclei at a final dilution of ~1,000 cells/μl was loaded onto the cartridge following the manual. The individual transcriptomes of encapsulated cells were barcoded during RT step and resulting cDNA purified with DynaBeads followed by amplification per manual guidelines. Next, PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to the sequencing adapters and indexed by PCR. The indexed DNA libraries were double-size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina NovaSeq S4 platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher). Sequencing depth was ~200 million reads per sample on average. FASQ files were processed using SEQC pipeline 36 for quality control, mapping to GRCH38 reference genome, and log2 transformation of the data with the default SEQC parameters to obtain the gene-cell count matrix.