title: GSM7968659: iso_spleen; Mus musculus; RNA-Seq
library_construction_protocol: Cardiac allografts normally arrest at day 7 after transplantation because of acute transplantation rejection. Therefore, we harvested spleens and cardiac allografts of allograft group recipients at day 6 before cardiac allografts arrest. At the same time, spleens and cardiac isografts of isograft group recipients were harvested. CD45+ cells were respectively isolated from cardiac allografts, allograft group recipients' spleen, cardiac isografts and isograft group recipients' spleen after digestion, centrifugation and flow sorting. In brief, cardiac grafts were cut into pieces and digested with 1 mg/mL of collagenase B (Roche 11088815001) in HEPES buffer for 45 min at 37°C. After digestion, cell suspensions were collected and filtered with 70-μm cell strainers (BIOFIL), Percoll (Beijing Solarbio Science & Technology Co., Ltd.) was used to purify mononuclear cells through density centrifugation with 600g 25min RT not-brake. Splenocytes were prepared through grinding, erythrocyte lysis, washing and centrifugation (600g, 3min). CD45+ live cells were then sorted by FACSAria flow cytometer after Cardiac graft infiltrating cells and splenocytes stained with CD45-APC (Biolegend 103112) and Zombie GreenTM Fixable Viability Kit (Biolegend 423112). Single cell Gel Bead-In-Emulsions (GEMs) were constructed using Chromium Single Cell 5' Library and Gel Bead Kit following the manufacturer's instruction. Briefly, Briefly, FACS-sorted cells were washed with 0.04% BSA PBS for three times and were resuscitated to a concentration of 700~1200 cells/uL (Viability≥85%) as determined by Countess™ II Automated Cell Counter. Cells were captured in droplets at a targeted cell recovery of cells. Single cell GEMs was consisted of single cell and specific 16nt 10× Barcodes, 10nt unique molecular identifiers (UMI). In single cell GEMs, tagged cells lysed and released mRNA was barcoded in GEMs. Reverse transcription was performed in GEMs. After the RT step, the amplification of 10× Barcoded cDNA was completed after emulsion broken. Amplified cDNA was then used for both 5' gene expression library construction and TCR enrichment. Library was sequenced on NovaSeq platform (Illumia). Sequencing depth was set to be 30 million per cell.