GSM8395992: FUS CAR; Homo sapiens; RNA-Seq
In the CD19 CAR-T cancer model, mice blood was collected from  tail vein at day 43 post CAR-T cell treatment. Mice peripheral blood  mononuclear cell (mPBMC) was isolated after red blood cells were  lysed. Anti-human CD3 and CD45 antibodies were used to identify T cell population. T cells freshly sorted from mPBMC were subjected to RNA-seq library preparation by Chromium next GEM single cell 3' reagent kits v3.1 (dual index). Briefly, in step 1, after preparing fresh master mix, single cell suspension  (10,000 cells per sample), and gel beads, GEMs were generated by  combining the prepared master mix containing cells, gel beads and  partitioning oil onto chromium next GEM chip G in the chromium controller.  Immediately after GEM generation, GEMs were transferred to PCR tubes  to run GEM-RT incubation. In step 2, post GEM RT cleanup was performed with dynabeads, which was followed by cDNA amplification and cDNA cleanup with SPRlselect. The purified cDNA was quantified using BioAnalyser. In step 3, cDNA amplicon size was further optimized by enzymatic fragmentation and size selection. P5, P7, i7 and i5 sample indexes, and TruSeq read 2 were added via end pair, A tailing, adaptor ligation and PCR. A post-library-construction QC was run. In step 4, the chromium single cell 3' gene expression dual index library containing standard illumina paired-end constructs which begin and end with P5 and P7 were sequenced by a Hiseq 2500 RapidRun. The 16bp 10x Barcode and 12bp UMI were encoded in read 1, and read 2 is used to sequence the cDNA fragment. I7 and i5 index sequences were incorporated as the sample index reads.