title: GSM8946968: Control_2; Mus musculus; RNA-Seq
library_name: GSM8946968
library_construction_protocol: After euthanasia, hearts were minced with a razor blade and digested with a combination of collagenase I (3.6 mg/ml), DNase I (60 U/ml), and hyaluronidase (60 U/ml) for 1 hour at 37C. Red blood cells were lysed and cells were resuspended in 1 ml PBS containing 1% BSA with Fc blocking solution for 30 minutes on ice. Cells were stained for 30 minutes at room temperature with primary antibodies listed in Supplemental data. After staining for 30 minutes, cells were spun, washed with 1% BSA, and sorted using a Becton–Dickenson FACSAriaII. For scRNA-seq studies, we sorted ~25,000 live, CD45+ cells into individual BSA coated tubes (see Figure 2 for gating strategy used for scRNAseq experiments). Cells were initially counted with trypan blue to ensure quality of cells after sorting. After reaching the core, cells were counted with a BioRad TC20 Automated Cell Counter (PN 1450102) and approximately 5,000 cells from each animal were subjected to bead emulsion and 10X genomics. This was done for N=2-3 animals from each experimental group (Control, HFpEF + sham, HFpEF + tVNS). 10xChromium single cell libraries were prepared according to the standard protocol outlined in the manual. Briefly, sorted single cell suspension, 10x barcoded gel beads, and oil were loaded into Chromium™ Single Cell Chip G to capture single cells in nanoliter-scale oil droplets by Chromium™ Controller and to generate Gel Bead-In-EMulsions (GEMs). We used “Chromium™ Single Cell 3' GEM, Library & Gel Bead Kit v3.1” and chip G for these experiments.  Full length cDNA libraries were prepared by incubation of GEMs in a thermocycler machine. GEMs containing cDNAs were broken and all single cell cDNA libraries were pooled together, cleaned using DynaBeads MyOne™ Silane beads (Fisher PN 37002D), and pre-amplified by PCR to generate sufficient mass for sequencing library construction. Sequencing libraries were constructed by following the steps: cDNA fragmentation, end repair & A-tailing, size selection by SPRIselect beads (Beckman Coulter, PN B23318), adaptor ligation, sample index PCR amplification, and a repeat of SPRIselect beads size selection. The final constructed single cell libraries were sequenced by Illumina Nextseq2000 machine with total reads per cell targeted for a minimum of 25,000.
identifiers.namespace: GEO
identifiers.id: GSM8946968_r1