GSM5285709: PBMCs from healthy control HIP015_PBMC_2; Homo sapiens; RNA-Seq
Immediately after Ficoll separation, 50,000 PBMCs were resuspended in RPMI + 10% FCS at a concentration of 75,000 cells/mL. 200 μL of this cell suspension (15,000 cells) was then loaded onto Seq-Well arrays pre-loaded with mRNA capture beads (ChemGenes). Following four washes with DPBS to remove serum, the arrays were sealed with a polycarbonate membrane (pore size of 0.01 µm) for 30 minutes at 37°C and then frozen at -80°C for no less than 24 hours and no more than 14 days to allow batching of samples processed at irregular hours. Next, arrays were thawed, cells lysed, transcripts hybridized to the mRNA capture beads, and beads recovered from the arrays and pooled for downstream processing. Library preparation was performed with a Nextera XT DNA library preparation kit (Illumina FC-131-1096) with 1 ng of pooled library using dual-index primers. Tagmented and amplified libraries were again purified by Agencourt AMPure XP beads with a 0.6x volume wash followed by a 1.0x volume wash, and quality and concentration determined by Fragment Analysis. Libraries between 400-1000bp with no primer peaks were considered successful and pooled for sequencing. Sequencing was performed on a NovaSeq S2 instrument (Illumina; Chan Zuckerberg Biohub). The read structure was paired-end with read 1 beginning from a custom read 1 primer11 containing a 12bp cell barcode and an 8 bp unique molecular identifier (UMI), and with read 2 containing 50bp of mRNA sequence. 