GSM8067207: Sample Spleen, scRNAseq; Gallus gallus; RNA-Seq
Spleen of 3 weeks-old wild-tpye and RAG1 knockout chicken were freshly collected and washed with Phosphate Buffered Saline (PBS without Ca2+/Mg2+, pH 7.0-7.2). The tissue was mechanically digested with blade and chemically digested with 0.05% trypsin-EDTA diluted in Hank's Balanced Salt Solution (HBSS, without Mg2+/Ca2+, pH 7.2) for 10 minutes at 37°C. The 0.05% trypsin-EDTA was inactivated by filtered Fetal Bovine Serum (FBS) with 10% volume of trpysin-EDTA. The red blod cells were removed using Red Blood Cell Lysis Solution (Miltenyil Biotech) and filtered out by 40μm cell strianer. 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 10,000. 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 and enriched 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 (Agilent technologies).  And then the libraries were sequenced using HiSeq platform(Illumina) according to the read length in the user guide.