title: GSM7162713: 6H post HSV2 infection, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Human palatine tonsillar tissue was obtained from a patient undergoing elective tonsillectomy, placed immediately into 50ml conical tubes containing 30ml RPMI 1640 supplemented with 10% (v/v) fetal bovine serum; 100U/ml penicillin, 100ug/ml streptomycin, 0.25ug/ml Amphotericin B (Gibco Cat. no. 15240062), and 5ug/ml Gentamicin (Lonza Cat. no. 17-518L) (RPMI-t) and processed within two hours. To isolate CD4+ T cells, tonsillar tissue was cut into small sections using sterile scissors and manually dissociated by passing through a 70mm mesh strainer to form single cell suspensions. The single cell suspensions were then pelleted and washed 2x in ice cold RPMI-t. Cells were resuspended in 45ml RPMI-t and overlayed on 15ml Ficoll and centrifuged at 1200xg for 20 minutes. Cells isolated from the interphase were washed twice with RPMI-t and CD4 T cells were isolated by negative selection using the EasySepTM Human CD4+ T Cell Enrichment Kit (Cat. 19052, STEMCELL Technologies). Cells isolated from tonsil were cultured in RPMI-t,  harvested at 0 (mock), 6 and 24 h post-infection and dead cells were depleted by FACS. Single cell libraries were prepared at the Yale Center for Genome Analysis. Gel/bead emulsions were prepared by loading single cell suspensions in RT Master Mix on the Single Cell A Chip with a pool of 750,000 barcoded gel beads each containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence. Leftover biochemical reagents and primers were removed using silane magnetic beads. Full-length, barcoded cDNA was then amplified by PCR to generate sufficient mass for library construction. Enzymatic Fragmentation and Size Selection were used to optimize the cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) was added during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.