GSM9666487: scRNA-seq of P1; Mus musculus; RNA-Seq
Samples were minced with a sterile scalpel into 1 mm3 fragments, suspended in 5ml of digestion buffer consisting of 2 mg/mL Collagenase type I (Sigma，C0130)，2 mg/mL Collagenase type II (Sigma, C6885) and 200U/ml DNase I (Worthington, LS006344) in RPMI medium (Coring,10-040-CV), and incubated in 37℃ water bath with shaking for 60 min. The suspension was passed through a 100 μm filter (Falcon,3523260) and centrifugated (400g, 10 min, 4℃). Pelleted cells were resuspended in red blood cell lysis buffer (Solarbio,R1010), incubated for 2 min, passed through a 40 μm filter (Falcon,3523240), collected by centrifugation (400g, 10 min, 4℃) and resuspended in PBS (BI, 02-024-1ACS)  containing 0.04% BSA (Sigma,B2064). Cells were manually counted by Trypan blue (Thermo, T10282) and AO-PI (LUNA, D23001) after each centrifugation (400g, 10 min, 4℃) and resuspended. Single-cell libraries were constructed with the DNBelab C Series Single-Cell Library Prep Kit (MGI, Cat. # 940-001818-00). Briefly, single-cell suspensions were subjected to droplet generation, emulsion breakage, bead collection, reverse transcription, and cDNA amplification to generate barcoded libraries. Library quantification and quality control were performed using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Q33216) and an Agilent 4200 TapeStation (Agilent, G2991AA), respectively. Finally, DNA nanoballs (DNBs) were loaded into patterned nanoarrays and sequenced on a DNBSEQ platform. The sequencing run was configured with the following read lengths: 30 bp for read 1, inclusive of 10 bp cell barcode 1, 10 bp cell barcode 2, and 10 bp unique molecular identifier (UMI), 100 bp of transcript sequence for read 2, and 10 bp for sample index.