Human single nuclear RNA seq PoPH and non-PoPH Cirrhosis Liver
snRNAseq data from PoPH (N=4) and non-PoPH cirrhosis ("Control", N=5) human adult liver tissue. Tissue was snap frozen during liver transplant operation, cold enzymatic digestion into single nuclei suspension (using modified Humphrey's Kidney protocol), and subjected to snRNAseq using 10X Chromium platform. H5 files are provided, which have been generated from FASTQ files using the CellRanger cloud platform.
Steps to reproduce
Explanted human liver tissue was obtained at the time of liver transplantation. After removal of the capsule, multiple samples of 5-mm cubed segments of tissue were obtained from different lobes of the explant and snap frozen in liquid nitrogen within 30 minutes of operative removal from the patient. Single nucleus suspensions were then generated using a modification of the “Humphrey’s” protocol for human kidney tissue (https://www.protocols.io/view/nuclei-isolation-from-human-kidney-for-single-nucl-81wgbobnlpko/v1) Nuclear lysis buffer 0 (NLB0), buffer 1 (NLB1), and buffer 2 (NLB2) were created Nuclei EZ lysis buffer (Sigma Aldrich, St Louis, MO, USA), cOmplete ULTRA protease inhibitor (Sigma Aldrich, St Louis, MO, USA), Promega RNAsin Plus (Fisher Scientific, Vilnius, Lithuania), and Invitrogen SUPERaseIN (Fisher Scientific, Vilnius, Lithuania), suspended in 0.1% Bovine Serum Albumin solution. Briefly, liver tissue cubes were placed on a 60mm dish, 1ml of NLB1 was added, and tissue was mechanically dissociated via mincing with a fresh razor blade. After transfer to a Dounce tissue grinder over ice, and addition of 1ml of NLB1, further mechanical dissociation was performed via grinding 15-20 times with a loose pestle. The homogenate was passed through a 200ul strainer and collected in a 50ml conical tube. The homogenate was returned to a Dounce tissue grinder, and ground 7-10 times with a tight pestle. Following this, 2ml of NLB1 was added, and the solution was incubated over ice for 5 minutes. The homogenate was passed through a 40um strainer into a 50ml conical tube, then transferred to a 15ml conical tube. This tube was centrifuged at 500G for 5 minutes at 4 degrees Celsius. After discarding the supernatant, the resulting pellet was suspended in 4ml of NLB2 and incubated on ice for 5 minutes. The tube was again centrifuged at 500G for 5 minutes at 4 degrees Celsius. After again discarding the supernatant, the pellet was resuspended in 2ml of NSB. The resulting suspension was passed through a 10um filter (pluriSelect Life Sciences, Leipzig, Germany) into a 50ml conical tube. Single nucleus samples were prepared per instructions provided in the 10X Genomics Single-Cell 3’v3.1 Reagent Kit. For each sample, nuclei concentration was adjusted to 700-1200 nuclei/ul, 11200 nuclei were loaded into the 10X Chromium controller for 10X single nuclei sequencing (targeting recovery of 7000 nuclei), and a Gel Beads in Emulsion was generated. cDNA libraries were prepared as per instructions provided in the 10X Genomics Single-Cell 3’v3.1 Reagent kit, 18 cycles were used for cDNA amplification, and single nuclear libraries were sequenced using Illumina NovaSeq 6000. Single nuclear data were processed using the 10X Cell Ranger software version 6.1, mapping reads to a modified GRCh38 human genome which included intronic regions to ensure quantification of reads derived from immature messenger RNA (mRNA) present in the nucleus.