GSM8853600: Handling_3h_S14; Mus musculus; ATAC-seq
Nuclei isolation was performed similarly as for the bulk ATAC-Seq, with the addition of RNase inhibitors as previously described (Grandi et al., 2022). All steps were performed on ice. Briefly, one vHC was homogenized in lysis buffer (26 mM Sucrose, 30 mM KCl, 10 mM MgCl2, 20 mM Tricine-KOH (pH 7.8), 1 mM DTT, 0.5 mM Spermidine, 0.15 mM Spermine, 0.3% NP-40, Complete protease inhibitor, 0.2 U/μl RNAse inhibitor in nuclease-free water) by 10 strokes with pestle A and 20 strokes with pestle B. The sample was filtered through a 70 μm Flowmi strainer and centrifuged for 5 min at 400 g at 4°C. Supernatant was removed and additional lysis buffer was added to resuspend the pellet. The sample was then mixed with 50% Iodixanol solution (30 mM KCl, 26 mM Sucrose, 10 mM MgCl2, 20mM Tricine-KOH (pH 7.8), 50% Iodixanol solution in nuclease-free water) and underlaid by 30% Iodixanol solution (50% Iodixanol solution in lysis buffer) and 40% Iodixanol (50% Iodixanol solution in lysis buffer) layers. The sample was centrifuged for 20 min at 3000 g at 4°C with brakes off. The nuclei band was collected and mixed with ATAC buffer (10 mM NaCl, 10 mMTris-HCl (pH7.5), 3 mM MgCl2, 0.1% Tween20, 0.2 U/μl RNase inhibitor in nuclease-free water) before the nuclei count was assessed. The sample was centrifuged for 5 min at 500g at 4°C and supernatant was removed. Diluted nuclei buffer (0.1 M DTT, 1 U/μl RNase inhibitor, 1X Nuclei buffer (10X) in nuclease-free water) was added to achieve the desired nuclei concentration. 10X Multiome libraries were prepared following the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide (CG000338, Rev. F, 10X Genomics) with a targeted nuclei recovery of 10'000 nuclei (1 sample failed due to clogging). 7 cycles were performed for sample index PCR during ATAC library preparation. 6 cycles were used for cDNA amplification and a total of 13 cycles was performed for sample index PCR for gene expression library preparation. RNA integrity was assessed on an Agilent Tapestation, according to the manufacturer's protocol. Samples of each library type were pooled. Both libraries (gene expressions and ATAC) were then sequenced on one Illumina X Plus 25B flow cell on individual lanes with an adjusted read configuration (150-10-24-150 bp). ATAC-seq reads were trimmed to 49nt for further processing.