GSM7466599: LNCaP Control, replicate 2, scRNAseq; Homo sapiens; RNA-Seq
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 concentrationof cellular suspensions wasadjustedto1 × 104cellsper μL 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 4°C. 10 µl purified cDNA sample, 5 µl Fragmentation Buffer, 10 µl Fragmentation Enzyme, and 25 µl Buffer EB were mixed and then processed in a program having 10 cycles of 32°C for 5 min, 65°C for 30 min, and hold at 4˚C. 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 µl sample, 20 µl Ligation Buffer, 10 µl DNA Ligase, and 20 µl Adaptor Oligos. The thermal cycler was 20°C for 15 min and hold at 4°C. 0.8X SPRI was used to do adaptor ligation selection. A PCR reaction with a mix containing 30 µl sample, 10 µl SI Primer, and 50 µl AmpMix was used to add sample index. The mix was incubated in a thermal cycler with the following protocol: 98°C for 45 sec; 5 cycles of 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec; 72°C for 1 min; hold at 4°C. 0.6X SPRI and 0.8X SPRI were used to do double sided size selection. Finally, fragments containing R1, R2, P5, P7, and sample index were produced. 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.