GSM9377234: Donor_5,PBMC,CD4,PMAiIL2,rep1; Homo sapiens; RNA-Seq
PBMCs were isolated by Ficoll gradient centrifugation with ammonium-chloride-potassium buffer-based red-blood cell lysis and viably frozen in liquid nitrogen. Intra-epithelial lymphocytes (IEL) and Lamina propria-resident immune cells (LP cells) were isolated in the method of Trapecar et al. DOI:10.1089/aid.2017.0208 and pooled after isolation. Epithelial stripping was performed in an EDTA+DTT buffer with mechanical disruption on a gentleMACS dissociator, with IEL cells liberated to the supernatant. The remaining lamina propria fraction was digested in CLSPA+DNAse buffer and subjected to additional gentleMACS dissociation and mechanical disruption by syringe. LP cells were isolated by filtration of the supernatant through a 70 micron filter. LP and IEL cells were treated with DNAse, filtered through a 40 micron filter, and rinsed in complete RPMI culture media. Cells were then subjected to a dead cell removal protocol (Biolegend) to remove epithelial debris. scRNAseq libraries were prepared according to manufacturer instructions (10x Genomics scRNAseq v3.1 3' sequencing, catalog 1000269, protocol ). Gel-bead and cell emulsions (GEMs) were generated using the Chromium controller, subjected to reverse transcription, and first-strand cDNA was extracted from GEMs. Purified cDNA was PCR pre-amplified (12-cycles, 1-minute extension) with primers binding the P7 region and the Template Switching Oligo, cleaned up by solid-phase reversible immobilization (SPRI) beads, analyzed by HS DNA 5000 capillary electrophoresis (Agilent), and concentration calculated by Qubit (ThermoFisher).  From this material, 25% was used for Illumina NGS library construction involving fragmentation, end-repair/A-tailing, adapter TA ligation, and SPRI double-sided size selection to remove adapters and unfragmented products.  Libraries were amplified with Illumina indexing/sequencing i5 and i7 primers (13 cycles), then cleaned with SPRI beads before being quantified by Qubit and the fragment size determined using a Tapestation (Agilent).  Libraries were then pooled at an equimolar concentration and sequencing was performed using NovaSeq or NovaseqX sequencing (Illumina). CITE seq was performed as above, but with the following additional steps. The TotalSeq-B Universal Human antibody panel (Biolegend, catalog 399904) of lyophilized DNA-tagged antibodies was resuspended and spun down according to manufacturer's instructions.  Meanwhile, cells were treated with Human TruStain FcX (Biolegend, catalog 422301) to prevent antibody binding by human Fc receptors.  Next, cells from each donor were stained with Totalseq-B hash-tag oligonucleotide antibodies (Biolegend, catalog 155831, 155833, and 155835) then added to three pools comprising untreated PBMCs, untreated CD4 cells, and PMA/ionomycin stimulated CD4 T cells. Pooled samples were stained in parallel with the TotalSeq-B Universal Human antibody panel, washed, and prepared for scRNA seq library construction as above but with additional PCR amplification of cell surface feature libraries following manufacturer instructions (10x Genomics protocol #CG000206).