title: GSM9583107: cy0692, M2/M4, control; Macaca fascicularis; RNA-Seq
library_construction_protocol: PBMC from Mauritian-origin cynomolgus and Indian-origin rhesus macaques were isolated from fresh whole blood by Ficoll-Plaque centrifugation and transported immediately to the University of Wisconsin Biotech Center for 5' RNAseq analysis Chromium GEX libraries were generated according to the manufacter's instructions for 5' single cell RNAseq (v2 NextGEM User Guide Rev.E). Briefly, PBMC were resuspended in master mix and loaded together with partioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was reverse transcripted to full-length cDNA, which contains an Illumina R1 primer sequence, a 16 nt 10X Barcode, a 10 nt unique molecular identifier and a 13 nt template switch oligo. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR, enzymatically fragmented and appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During library construction An Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences plus i5 and i7 sample index sequences were added during library construction. The resulting templates from two animals were sequenced on a NovaSeq X+ instrument with a 1.5B flow cell. A modified sequencing protocol was used with 29 cycles for R1 to collect 10X barcode/UMI sequences, followed by 289 cycles for R2. This protocol maximized the amount of sequence data from the highly polymorphic 5' end of each cDNA molecule so that transcripts from closely related MHC alleles could be resolved.