title: GSM8486210: WT-2-10XTCR; Mus musculus; OTHER
library_construction_protocol: The tissue was placed in a petri dish on ice and cut into small pieces of 2-4 mm. The pieces were infused with the RPMI/enzyme mix (Miltenyi Biotec), transferred to a gentleMACS C tube containing RPMI/enzyme mix, attached to the sleeve of the gentleMACS Octo Dissociator and run program. After termination of the program, the suspension was passed through a 70um straine and spun down at 300 x g for 10 min at 4 °C, removed the supernatant and added 10 times volume 1 x Red Blood CellLysis Solution(130-094-183, Miltenyi Biotec) to lytic red blood cells in the refrigerator at 4 °C for 10 min, then added 10 ml 1 x PBS+0.04 % BSA and the resulting suspension was centrifuged at 300 x g for 10 min. Library was performed accordimg to the manufacter's instructions (single cell 5'v2 protocol. 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). Immediately following GEM generation, the Gel Bead is dissolved, primers are released, and any co- partitioned cell is lysed. Primer than contains an Illumina TruSeq Read 1 (Read 1 sequencing primer), 16 nt 10x Barcode, 12 nt unique molecular identifier (UMI) and 30 nt poly(dT) sequence are mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces Barcoded, fulllength cDNA from poly-adenylated mRNA.After incubation, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library construction.Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. TruSeq Read 1 (Read 1 primer sequence) is added to the molecules during GEM incubation. P5, P7, a sample index, and TruSeq Read 2 (Read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification. Qubit HS quantification, Agilent 2100 Bioanalyzer/Fragment Analyzer 5300 quality control, the final library size of about 450bp.