C57BL/6J, HBOC, replicate 2, scRNAseq
Single-cell sequencing was performed. The cells were digested with 0.25% (w/v) trypsin (Gibco) and collected by centrifugation for subsequent analysis. Samples with 90% living cells were analyzed.The concentration of cellular suspensions was adjusted to 10000 cells per L using PBS containing 0.04% bovine serum albumin (BSA) (Thermo Fisher Scientific). The experiment was performed according to the manufacture of Chromium Next GEM Single Cell 3 Reagent Kits v3.1 (10x Genomics, PN-1000121). The cDNA was digested into 200-300 bp fragments by PCR at first. Before PCR proceeded, the thermal cycler was pre-cooled at 4C. 10 l purified cDNA sample, 5 l Fragmentation Buffer, 10 l Fragmentation Enzyme, and 25 l Buffer EB were mixed and then processed in a program having 10 cycles of 32C for 5 min, 65C for 30 min, and hold at 4C. SPRIselect Reagent Kit (0.6X SPRI and 0.8X SPRI) were used to do double sided size selection of post fragmentation, end-repair, and A-tailing. Then, a PCR reaction was set up to perform adaptor ligation with a system of 50 l sample, 20 l Ligation Buffer, 10 l DNA Ligase, and 20 l Adaptor Oligos. The thermal cycler was 20C for 15 min and hold at 4C. 0.8X SPRI was used to do adaptor ligation selection. A PCR reaction with a mix containing 30 l sample, 10 l SI Primer, and 50 l AmpMix was used to add sample index. The mix was incubated in a thermal cycler with the following protocol: 98C for 45 sec; 5 cycles of 98C for 20 sec, 54C for 30 sec, 72C for 20 sec; 72C for 1 min; hold at 4C. 0.6X SPRI and 0.8X SPRI were used to do double sided size selection. The insert size of the library was examined using Agilent 2100. The effective concentration of the library was examined by preliminary quantitative Q-PCR performed by Qubit3.0 to accurately quantify.