GSM9108990: IBD; Homo sapiens; RNA-Seq
Human CD4⁺ T cells were isolated from spleen, and colon tissues of humanized mice. Tissues were dissociated, passed through a 70 μm cell strainer, and red blood cells were lysed using RBC lysis buffer (eBioscience). CD4⁺ T cells were purified by MACS (Miltenyi Biotec) according to the manufacturer's protocol. Use LUNA-FL™ Automated Fluorescence Cell Counter (logos biosystems) to consult the 10x Genomics Single Cell Protocols Cell Preparation Guide and the Guidelines for Optimal Sample Preparation flowchart (Documents CG00053 and CG000126 respectively) for more information on preparing cells. Libraries were prepared using the Chromium controller according to the 10x chromium Next GEM Single Cell 3' v3.1 protocol(CG000315). Briefly, the cell suspensions were diluted in nuclease free  water to achieve a targeted cell count of 15,000. Cell suspension was mixed with master mix and  loaded with Single Cell 3'V3.1 Gel Beads and Partitioning Oil into a chromium Next GEM chip G. RNA transcripts from single cells were uniquely barcoded and reverse transcribed within droplets. cDNA molecules were pooled and the cDNA pool then go through an end repair process, the  addition of a single 'A' base, and then ligation of the adapters. The products are then purified andenriched with PCR to create the final cDNA library. The purified libraries were quantified using    qPCR according to the qPCR Quantification Protocol Guide(KAPA) and qualified using the Agilent Technologies 4200 TapeStation . And then the libraries were sequenced using NovaSeq platform (Illumina) according to the read length in the user guide.