title: GSM5419818: BILE a; Mus musculus; RNA-Seq
library_construction_protocol: When sacrifice, mouse was anesthetized using phenobarbital. Upon opening the abdomen, the gallbladder was exposed and cystic duct was ligated. The gallbladder was soon removed avoiding liver tissues at the gallbladder bed. After aspiration of the bile, the gallbladder was washed with D-hanks solution. Thereafter, the gallbladder was cut into pieces and put in 1.5ml Trypsin-EDTA (0.25%) solution (No.25200-056, Gibco®) and incubated at 37°C for 20 minutes and then centrifuged at 1500rpm for 5 minutes. After removing the supernatant, the pellet was suspended in Trypsin-EDTA solution and repeated the digestion process additionally for twice. The collected cell pellets were then suspended in Gibco® RPMI 1640 medium. The living singles cells were then picked by a mouth pipette and transferred to cold scRNA-seq lysis buffer.For the quality check and counting of single cell suspension, the cell survival rate was > 80%. The cells that had passed the test were washed and re-suspended to prepare a suitable cell concentration of ~1,000 cells/ul for 10x Genomics ChromiumTM. The loaded Single Cell 3' Chip was placed on a 10X Genomics Chromium Controller Instrument (10X Genomics, Pleasanton, CA, USA) to generate single cell gel beads in emulsion (GEMs). GEMs were constructed for single cell separation according to the number of cells to be harvested. After GEMs were normally formed, GEMs were collected for reverse transcription in a PCR machine for labeling. The GEMs were oil-treated, and the amplified cDNA was purified by magnetic beads, and then subjected to cDNA amplification and quality inspection. The 3ʹ Gene Expression Library was constructed with the quality-qualified cDNA. After fragmentation, adaptor ligation, sample index PCR, etc., the library is finally quantitatively examined. Single cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Library & Cell Bead Kit (Cat. No. 120237, 120236, 120262; 10X Genomics) according to the manufacturer's protocol. The final library pool was sequenced on the Illumina instrument using 150-base-pair paired-end reads.