title: GSM4087819: HCCHepABC [Single-cell]; Danio rerio; RNA-Seq
library_construction_protocol: Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis (HCC or no HCC). The remaining half of each liver was dissociated into single-cell suspensions and prepared for single-tube single-cell RNA sequencing (scRNA-seq) based on the 10X Genomics platform. Dissected liver tissue was immersed in 5% Fetal Bovine Serum (FBS) in Hank's Buffered Saline Solution (HBSS) without phenol red, with calcium and magnesium and chopped finely. The cells were were homogenized in 1 mL of 0.25% trypsin + EDTA for 5 minutes at room temperature and re-suspended in 1 mL of 5%FBS+5mM EDTA in HBSS-Free solution. Cells were then filtered through a 40 micron membrane filter, and the filtrate was centrifuged at 1200 RPM, 4C for 5 minutes. The cell pellet was then re-suspended in phosphate buffered saline with 0.04% bovine serum albumin.  The cell suspension was then filtered again through 40 micron cell strainers to obtain a liver single-cell suspension. Viability and cell count were assessed on Countess I (Thermo Scientific).  The Chromium Single Cell Gene Expression Solution with 3' chemistry, version 2 (PN-120237) was used to barcode individual cells with 16 bp 10x Barcode and to tag cell specific transcript molecules with 10 bp Unique Molecular Identifier (UMI) according to the manufactures instruction.  Equilibrium to targeted cell recovery of 6,000 cells along with 10x Gel Beads and reverse transcription reagents were loaded to Chromium Single Cell A Chip (PN-120236) to form Gel-Bead-In EMulsions (GEMs), the micro-droplets.  Within individual GEMs, cDNA generated from captured and barcoded mRNA was synthesized by reverse transcription at the setting of 53°C for 45 min followed by 85°C for 5 min.  Subsequent A tailing, end repair, adaptor ligation and sample indexing was performed in bulk according to the manufacturer's instructions. Multiple libraries were then normalized and sequenced on NovaSeq 6000 with 2x150 PE mode. Single-cell 3' RNA-Sequencing
attributes.tag: GEO Accession
attributes.value: GSM4087819