title: GSM8798524: liver cells from adult offspring with intrauterine hyperglycemia mother; Mus musculus; RNA-Seq
library_name: GSM8798524
library_construction_protocol: Fetal male offspring at E18.5 were obtained by caesarean section. Adult male offspring at 8 w were sacrificed for liver scRNA-seq after portal vein perfusion. Samples were pooled from three pregnant mice and adult individuals, respectively. Mice livers were digested in a mixed enzyme system containing collagenase IV (1 mg/ml; Worthington), collagenase I (0.5 mg/ml;Worthington), DNase I (0.02 mg/ml; Yeasen), hyaluronidase (30 U/ml, Sigma-Aldrich), 0.6% bovine serum albumin (BSA; Sigma-Aldrich), and 5% fetal bovine serum (FBS; Gibco) at 37 ℃ for 30 min. Then, the digestive system was filtered and centrifuged at 300×g for 5 min. After discarding the upper fluid, 2 ml erythrocyte lysate was added to remove the erythrocytes within 5 min. The cracking reaction was terminated by cleaning resolusion. Centrifuged 300×g for 5 min, then resuspended and counted the liver cells. Single-cell libraries were generated using a GemCode single-cell instrument and a 10x Genomics Single Cell 3′ Library & Gel Bead Kit v2 and Chip Kit according to the kit manufacturer's protocol (Ou Yi Bio, Shanghai). Brieﬂy, liver cells were suspended in 0.04% BSA–PBS and then counted with a Countess® II Automated Cell Counter. The cell concentration was adjusted to the desired concentration of 1×106/mL. Approximately 8000 cells were added to each channel. Nanoliter-scale gel beads in emulsion (GEMs) containing barcode information were generated and then reverse transcribed in a C1000 Touch Thermal Cycler (Bio-Rad). PCR ampliﬁcation was performed using cDNA as a template. Subsequently, the ampliﬁed cDNA was fragmented into approximately 200–300 bp fragments by a Bioruptor sonication device, and the traditional second generation sequencing process was followed, including, for example, sequencing linker P5 and sequencing primer R1. Finally, the DNA library was ampliﬁed by PCR.
identifiers.namespace: GEO
identifiers.id: GSM8798524_r1