title: GSM9316363: SR006552_sbr_7254_Male_26; Mus musculus; RNA-Seq
library_construction_protocol: Single nuclear suspensions were produced from murine sham and SBR liver samples using modifications of a previously published protocol by Nadelmann et al. as detailed below, with modifications made to utilize our available tissue lysis equipment and best homogenize our tissue samples while preserving nuclear integrity. Liver samples frozen in RNAlater (Thermo Fisher Scientific, cat. no. AM7021) were removed from the -80°C freezer and thawed on ice. Tissue homogenizer block (ServiceBio; Hubei, China) was placed in -20°C freezer to be cooled for tissue processing. Four working solutions were made to use in our protocol, according to the recipes published by Nadelmann et al; nuclei isolation buffer 1 (NIM1), nuclei isolation buffer 2 (NIM2), homogenization buffer (HB), and storage buffer (SB). Thawed liver samples were kept on ice throughout the nuclei preparation. RNAlater was removed from tubes by pipette, and 1mL of HB was added to each sample tube. The cooled tissue homogenizer block was loaded with sample tubes and into the tissue lysis machine (ServiceBio; Hubei, China), which was run for one minute at 40 Hz. Each sample was assessed for adequate lysis and homogenization. Samples were placed on ice to allow for further lysis in HB for 5 minutes. Homogenized tissue was each filtered through 40-m cell strainer into 50mL tubes, then filtrates were transferred to 1.5mL Eppendorf tubes and centrifuged at 4°C for 2min at 2000g. Supernatant fluid was removed via pipette and the nuclei pellet was resuspended with 500 L of SB. Suspension was transferred and filtered into a fluorescence activated cell sorting (FACS) tube (35 um filter). An unstained, negative control was prepared by pipetting 100 L of filtered sample into a separate FACS sorting tube. 4',6-diamidino-2-phenylindole (DAPI) was added to the remaining solution, mixed by pipette, and placed on ice for 15min to allow for sufficient staining. 150 L of SB was pipetted individually into new 1.5mL nonstick RNase-free microcentrifuge tubes and placed on ice, to collect nuclei samples after FACS sorting. Samples were sorted on an Aria-3 cell sorter (BD biosciences; Franklin Lakes, NJ) into new 1.5mL nonstick RNase-free microcentrifuge tubes containing 150 L of SB and centrifuged at 4°C for 5min at 500g. Supernatant was removed slowly without disturbing nuclei. Nuclei pellet was resuspended with a volume of SB according to sort count from FACS to achieve a concentration of approximately 1.0 x 10^6 nuclei for each sample. Samples were centrifuged afterwards at 4C at 500g for 5 minutes, then resuspended in SB. For each sample, 2L of the nuclei suspension was mixed with 18uL Trypan blue (1:1) dilution in a new 1.5mL non-stick RNase-free microcentrifuge tube. 10uL of each sample was loaded into a Countess cell counting chamber slide alongside 10uL of unstained samples into adjacent wells on the slide. Nuclei were counted to confirm concentration between 8 x 10^5 and 1.2 x 10^6. Wells loaded without Trypan blue staining were visualized under microscopy to confirm nuclei presence without debris and intact nuclear morphology. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps.  Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads.  Samples were then run on a Bioanalyzer to determine the cDNA concentration.  GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration.  For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell Kit, 48 rxns (PN-1000120), and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used.  The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeqX instrument.  Normalized libraries were sequenced on a NovaSeqX Flow Cell using the 151x10x10x151 sequencing recipe according to manufacturer's protocol.  Read 1 was trimmed to the 10x Genomics recommendation of 28bp. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.