GSM8392937: KPC RNA; Mus musculus; RNA-Seq
PDAC tumors were harvested from two female KPC and two female AKPC mice and snap-frozen. 20-30 mg tissue samples were resuspended in 1ml of wash buffer (0.25 M sucrose, 25 mM KCI, 5 mM MgCl2, 10 mM Tris-HCI (pH 7.5), 1 mM DTT (Sigma, D9779), 1X protease inhibitor (Fischer, PIA32965), 1 U µl−1recombinant RNAsin (Promega, PAN2515), 0.1% IGEPAL (Sigma, I8896) in molecular biology water) and then homogenized using a Dounce homogenizer with 2 pestles. First, a loose pestle was used for 5-10 strokes to break the tissue into small pieces and then a tight pestle was used for 15-25 strokes to fully homogenize the sample into a uniform solution. Homogenized tissue was filtered through a 30 μm filter (CellTrics, Sysmex) and then centrifuged with a swinging-bucket centrifuge (100 rcf, 10 min, 4˚C, Eppendorf, 5920 R). Nuclei were resuspended in 1 ml of wash buffer (0.25 M sucrose, 25 mM KCI, 5 mM MgCl2, 10 mM Tris-HCI (pH 7.5), 1 mM DTT (Sigma, D9779), 1X protease inhibitor (Fischer, PIA32965), 1 U µl−1recombinant RNAsin (Promega, PAN2515) in molecular biology water) and centrifuged again (100 rcf, 10 min, 4˚C, Eppendorf, 5920 R). Next, nuclei were resuspended in 400 µl of sorting buffer (1X protease inhibitor (Fischer, PIA32965), 1 U µl−1recombinant RNAsin (Promega, PAN2515), 1% fatty acid free BSA (Proliant, 7500804), 2uM 7-AAD (Invitrogen, A1310) in PBS (Corning, 21-040-CV)) and incubated on ice for 10 minutes. 120,000 nuclei were sorted into a Lobind tube with 90 µl collection buffer (5 U µl−1recombinant RNAsin (Promega, PAN2515), 5% fatty acid free BSA (Prolian, 7500804) in PBS (Corning, 21-040-CV) using an SH800 cell sorter (Sony). The resulting nuclei suspension was centrifuged again (500 rcf, 5 min, 4˚C, Eppendorf, 5920 R) and resuspended in 100 µl of collection buffer (1% fatty acid free BSA (Proliant, 7500804), 1X protease inhibitor (Fischer, PIA32965), 10 mM Tris-HCI (pH 7.4), 0.1% Tween-20 (Sigma, P7949-100ML), 0.1% IGEPAL (Sigma, I8896), 0.01% digitonin (Promega, G9441), 1mM DTT (Sigma, D9779), 1 U µl−1recombinant RNAsin (Promega, PAN2515), 3 mM MgCl2 and 10 mM NaCl in molecular biology water). This suspension was incubated on ice for 1 minute and centrifuged again (500 rcf, 5 min, 4˚C, Eppendorf, 5920 R). The supernatant was carefully removed without disturbing the pellet and 10 µl of 1X Nuclei Buffer (10x Genomics) was used to gently resuspend the pellet. Nuclei were counted using a hemocytometer and approximately 20,000 nuclei were used as inputs for the tagmentation reaction. Nuclei from the two KPC mice were pooled together and nuclei from the two AKPC mice were also pooled together, for a total of 2 sets of libraries. Then single cell multiome ATAC and gene expression libraries were generated according to manufacturer instructions (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 10x Genomics, 1000283; Chromium Next GEM Chip J Single Cell Kit, 10x Genomics, 1000234; Dual Index Kit TT Set A, 10x Genomics, 1000215; Single Index Kit N Set A, 10x Genomics, 1000212). The following PCR cycles were used: 7 cycles for ATAC index PCR, 7 cycles for complementary DNA (cDNA) amplification, and 13–16 cycles for RNA index PCR. A Qubit fluorometer (Life Technologies) was used to quantify final libraries and a TapeStation (High Sensitivity D1000, Agilent) was used to check size distribution. Finally, libraries were sequenced on NovaSeq 6000 sequencers (Illumina) with the following read lengths (Read1 + Index1 + Index2 + Read2): ATAC (NovaSeq 6000), 50 + 8 + 24 + 50; ATAC (NextSeq 500 with custom recipe), 50 + 8 + 16 + 50; RNA (NextSeq 500, NovaSeq 6000), 28 + 10 + 10 + 90. Paired snRNA-seq and snATAC-seq