GSM9530683: E19.5 colon, maternal obesity group, sample2; Rattus norvegicus; RNA-Seq
The tissues were washed three times with Hank's balanced salt solution (HBSS) including 1 mM EDTA, 1 mM DTT, 100 IU/mL penicillin and 100 mg/mL streptomycin, 10mM HEPES. Then the tissues were sliced into small fragments roughly and digested with 0.5 mg/mL type IV collagenase (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), 0.005% pancreatic enzymes, 0.002% EDTA and 500μg/mL DNaseI (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 37℃ for 45-55 min. A 1:1 ratio of DMEM, 10% fetal bovine serum was added to stop the digestion. The cell suspension was passed through a 40-μm filter and isolated via centrifugation (300 g, 4℃, 5 min). Cells was resuspended in 3 mL of red blood cell lysate until no red and then was stopped by 3 mL 0.1%BSA. The cells were isolated via centrifugation (300 g, 4℃, 5 min) and washed with 0.1%BSA. After re-centrifugation, the single-cell suspension was obtained. The cell viability was more than 80% and the caking rate was less than 5% in the present study. Dissociated cells with cell concentration in the range of 700-1200 cells/μL were stored in 0.1%BSA on ice until loading on a Chromium Controller (10x Genomics). During library preparation, Gel Beads carrying Poly-T primers tagged with Cell Barcodes and Unique Molecular Identifiers (UMIs), along with cells, are co-encapsulated within "oil droplets" to form Gel Beads-in-Emulsion (GEMs). Within this reaction system, cells are lysed and reverse transcription is performed to generate full-length cDNA sequences. Subsequently, the cDNA is purified via emulsion breaking and amplified by PCR to increase yield. Following quality control, the cDNA is chemically fragmented into ~200–300 bp segments, which then undergo end repair, A-tailing, and size selection. P7 Adapter linkers are ligated, and sample indices are introduced via PCR, ultimately yielding the sequencing library after a final round of size selection.