title: GSM8680984: Cigarette Smoke AAA; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were isolated using 10x Genomics' Chromium Nuclei Isolation Kit (10x Genomics, cat. no. 1000493) lysis buffer with the debris removal substituted by the Miltenyi Anti-Nucleus beads. Frozen tissue pieces were weighed on dry ice before transferring into a petris dish. 500ul of Lysis buffer comprised of Lysis Reagent (10x Genomics, cat. no. 2000558), Reducing Agent B (10x Genomics, cat. no. 2000087), and Surfactant A (10x Genomics, cat. no. 2000559) was added to the frozen tissue on ice. The tissue was chopped into smaller pieces with tissue scissors and then transferred to a 2ml glass douncer (Millipore Sigma, cat. no D8938). 8 strokes with the loose pestle A was required to fully dissociate the tissue. The total lysis time was 5 minutes. After dissociation, 500ul of lysate was transferred to the Nuclei Isolation Column (10x Genomics, cat. no. 2000562) and spun at 16,000 rcf for 20 seconds at 4C to remove leftover tissue. Following the centrifugation, the sample was vortexed for 10 seconds at 3000 rpm. An aliquot from the lysate was used to calculate the nuclei numbers using a 1:1 dilution with AOPI. This count was used to calculate the volume of buffer to resuspend the nuclei. The sample was spun at 500 rcf for 3 min at 4C. The supernatant was removed and the nuclei pellet was resuspended in Nuclei Separation buffer composed of Nuclei Extraction buffer (Miltenyi Biotec, cat. no. 130-128-024), PBS, BSA and Rnase Inhibitor (Millipore Sigma, cat. no. 3335402001). Miltenyi Anti-nucleus beads (Cat. No.130-132-997) were then added to the sample; 50ul for each million nuclei. The nuclei were incubated with the beads for 15 minutes on ice after which an additional 2ml of the Nuclei Separation buffer was added to the sample. Then the sample was passed through a LS column (Miltenyi Biotec, cat. no.130-042-401) attached to the MACS MultiStand (Miltenyi Biotec, cat. no. 130-042-303) with QuadroMACS separator (Miltenyi Biotec, cat. no. 130-042-302). The column was then washed twice with 1ml of the Nuclei Separation buffer. After the washes, the column was placed on a 15ml collection tube and 1ml of the Nuclei Separation buffer was added into the column and plunged into the tube. The purified nuclei were spun at 500 rcf for 5 minutes. The supernatant was removed and the nuclei pellet was washed with Wash Buffer comprised of 1x PBS, 10% BSA and RNase Inhibitor. The nuclei suspension was centrifuged at 4C for 5 min at 500 rcf and the nuclei pellet was resuspended in wash buffer. Nuclei counting was done using a 1:1 dilution with AOPI on a haemocytometer (Thermo Fisher, cat. no. 22-600-100). Following counting, the appropriate volume for each sample was calculated for a target capture of 20,000 cells and then the nuclei were loaded onto 10x 3' GEM X chip. After droplet generation, samples were transferred to a pre-chilled strip tube (USA Scientific, US14024700), and incubated in a Bio-Rad thermos cycler. Sample cDNA was recovered using Recovery Agent provided by 10x and subsequently cleaned up using a Silane DynaBead (Thermo Fisher) mix as outlined by the user guide Chromium GEM-X Single Cell 3' Reagent Kits v4. Purified cDNA was amplified for 11 cycles before being cleaned up using SPRIselect beads (Beckman Coulter). Samples were run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. cDNA libraries were prepared as outlined by the Chromium GEM-X Single Cell 3' Reagent Kits v4 user guide with modifications to the PCR cycles based on the calculated cDNA concentration. The molarity of each library was calculated based on library size as measured bioanalyzer (Agilent Technologies) and qPCR amplification data (Roche, cat. no. 07960140001). Gene Expression libraries were sequenced on Illumina's NovaSeqX with the following run parameters: read 1 – 28 cycles, read 2 – 90 cycles, index 1 – 10 cycles and index 2 – 10 cycles.