title: GSM5788544: Ntg_SHAM_2; Mus musculus; RNA-Seq
library_construction_protocol: For single cell RNA sequencing, isolated cardiac fibroblasts were subjected to magnetic bead sorting using a Miltenyi QuadroMACS magnetic cell separation kit. Briefly, cells were strained through a 70 µm cell strainer and resuspended in 180uL of sorting buffer (1XPBS, 0.5%BSA, 2µM EDTA) and 20µL of Cd11b microbeads. Samples were mixed well and incubated for 15 minutes at 4oC. Samples were washed through the magnetic columns and the flow through (containing the Cd11b- fraction) was collected. The cells were spun and resuspend in 160uL of sorting buffer with 40µL of MEFSK4 (Anti-feeder) microbeads. Cells were again incubated for 15 minutes at 4oC and sorted using magnetic columns. The flow through was discarded, and the magnetic columns containing the MEFSK4+ fraction were removed and rinsed with sorting buffer. Viability was assessed at this point for all preps. Viability was greater than 90% for all samples. Cells were lysed as previously described (Alexanian et al., 2021). Briefly cells were spun and rinsed in 1x HBSS with no FBS. Pellets were resuspended in 200uL of Lysis buffer (Tris-HCl 10mM pH 7.4, NaCl 10mM, MgCl2 3mM, Tween-20 0.1%, NP40 0.1%, Digitonin 0.01%, in Nuclease-free water), pipetted 10 times and kept on ice for 5 minutes. Nuclei were wash with 1 mL of wash buffer [Tris-HCl 10mM pH 7.4, NaCl 10mM, MgCl2 3mM, Tween-20 0.1%, in Nuclease-free water] inverting 3 times to mix. Nuclei were spun at 5000g for 3 minutes at 4oC. Nuclei were further washed in 1.2mL of 1X PBS with 1%BSA and filtered through a 30µm filter to remove any debris. Nuclei were counted using Trypan blue and a hemocytometer. Nuclei were resuspended at a concentration of 1000nuclei/µL in 1XPBS with 1%BSA with 8300 nuclei being loaded for subsequent 10X Genomics Chromium single cell RNAseq preparation. Single-cell droplet libraries were generated in the 10X Genomics Chromium controller according to the manufacturer's instructions in the Chromium Single Cell 3′ Reagent Kit v.3.1 with Dual Indexing User Guide. Additional components used for library preparation include the Chromium Single Cell 3′ Library and Gel Bead Kit v.3.1 (PN- 2000164) and the Chromium Single Cell 3′ Chip kit v.3.1 (PN-120236). Libraries were prepared according to the manufacturer's instructions using the Chromium Single Cell 3′ Library and Gel Bead Kit v.3.1 (PN- 2000164) and Chromium i7 Multiplex Kit (PN- 1000213). Final libraries were sequenced on the NextSeq 2000. All 14 samples were pooled and sequenced in one single lane. Sequencing parameters were selected according to the Chromium Single Cell v.3.1 specifications. Libraries were sequenced to a mean read depth of at least 20,000 total aligned reads per cell.