GSM5057800: 5R: Abx-02_d0_L2; Homo sapiens; RNA-Seq
Cryopreserved PBMCs were thawed and innate immune cell subsets were isolated using FACS. Within the live gated cells, CD14+ monocytes were identified as CD14+ (fraction A) while a mixture of the remaining monocyte and dendritic cell subsets was identified as CD14-CD56-HLA-DR+ (fraction B). After sorting and depending on the number of isolated cells, fraction A and B were mixed at a 2:1 ratio to yield a solution of monocytes and dendritic cells enriched for CD14- cells. FACS-purified cells were resuspended in PBS supplemented with 1% BSA (Miltenyi), and 0.5 U/μL RNase Inhibitor (Sigma Aldrich). About 9,000 cells were targeted for each experiment. Cells were mixed with the reverse transcription mix and subjected to partitioning along with the Chromium gel-beads using the 10X Chromium system to generate the Gel-Bead in Emulsions (GEMs) using the 3' V3 chemistry (10X Genomics). The RT reaction was conducted in the C1000 touch PCR instrument (BioRad). Barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was subjected to 0.6x SPRI beads cleanup (Beckman, B23318). 25% of the amplified cDNA was subjected to enzymatic fragmentation, end-repair, A tailing, adapter ligation and 10X specific sample indexing as per manufacturer's protocol. Libraries were quantified using Bioanalyzer (Agilent) analysis. Libraries were pooled and sequenced on an NovaSeq 6000 instrument (Illumina) using the recommended sequencing read lengths of 28 bp (Read 1), 8 bp (i7 Index Read), and 91 bp (Read 2).