title: GSM8806105: heart, control; Mus musculus; RNA-Seq
library_construction_protocol: Using aseptic, RNA-preserving conditions, control Db/db and T2D db/db mouse hearts were gravity-fed perfusion fixed under deep isoflurane anesthesia (3%) by ~15 seconds of a PBS flush, followed by ~15 seconds of 4% paraformaldehyde (PFA), and finished with 4% PFA with 60 mM KCl until cardiac arrest in diastole. Tissues were fixed for 24 hours in 4% PFA at 4°C and stored in 70% ethanol until paraffin embedding. Six 50 µm scrolls were cut from one control Db/db and one diabetic db/db formalin-fixed and paraffin-embedded (FFPE) heart blocks. Cells were isolated from scrolls by following the Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling protocol from 10X Genomics (CG000632 RevB) using the gentleMACS Octo Dissociator method. Approximately 200,000 isolated cells for each sample were used as input for the Chromium Fixed RNA Profiling kit from 10X Genomics (CG000477 RevD) according to the manufacturer's protocol. Briefly, cells were hybridized overnight with mouse BC001 probe set (Singleplex, ThermoFisher Scientific), followed by GEM generation and barcoding, and Illumina compatible library preparation. Libraries were sequenced on the Singular Genomics G4 benchtop sequencer; therefore, the Illumina libraries were converted using Singular Genomics conversion PCR primers. We targeted 25,000 read pairs per 10,000 cells for each sample and used a run recipe of: 28x10x10x50 (read1:i7index:i5index:read2).
identifiers.namespace: GEO
identifiers.id: GSM8806105_r1