GSM2525513: MIP1_CD45_4; Mus musculus; RNA-Seq
RNA was isolated with trizol according to manufacturer’s instructions with glycogen included as a carrier. Each sample was treated with DNase I (Roche) for 10 minutes and then RNA was further column purified with MinElute RNeasy kit (#28004, Qiagen). Following cell extraction, the non-myocyte fraction was resuspended in 100ul 5 mg/ml bovine serum albumin in PBS solution. Cell isolates were then incubated at 4°C for 20 minutes with 3 ug of CD90-APC (A14727, ThermoFisher), CD45-FITC (130-102-778, Miltenyi Biotec), CD31-BV421 (102423, BioLegend) and Podoplanin-PE/Cy7 (127411, BioLegned). The stained cells were washed in PBS/BSA solution and sorted using a BD FACS ARIA cell sorter. Each purified cell population was centrifuged at 1000xg for 5 minutes, cell media was aspirated and 1 ml Trizol was added to isolate RNA. Ribosomal RNA was depleted with Ribo Zero Gold (Illumina), RNA quality ascertained by Bioanalyser and cDNA generated with SuperScript II Reverse Transcriptase (ThermoFisher). Libraries were created with TruSeq Stranded Total RNA kits (Illumina) and read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer. Each sample contained ~45 million 50-bp single-end reads.