GSM3937566: 2168-1_heart_WT; Mus musculus; RNA-Seq
To obtain high quality total RNA, the hearts were processed first in the Trizol reagent (ThermoFisher Scientific, Catalog # 15596018), and then by RNeasy mini columns (Qiagen, catalog # 74104) as follows. To avoid sampling bias, the entire hearts were homogenized in Trizol reagent at a weight (mg) to volume (µL) ratio of 1:15. Then, 1 mL of the homogenate was processed following the manufacturer's protocol until the step of phase separation. 0.5 mL of the aqueous phase was then mixed with an equal volume of 70% ethanol for subsequent RNA purification by the RNeasy mini kit. An on-column DNAse (RNase-Free DNase Set, Qiagen catalog # 79254) treatment step was included. The concentration of the RNA was determined by Qubit fluorometric quantitation (Qubit RNA BR Assay Kit, ThermoFisher Scientific, catalog # Q10210). The integrity of the total RNA was determined by a Fragment Analyzer (Advanced Analytical Technologies, Inc). The average RNA Quality Number (RQN) was 8.77 ± 0.07 (Mean ± SEM, n=24). 1 µg of total RNA from each sample was used for RNA sequencing library preparation with the TruSeq® Stranded mRNA Library Prep kit (Illumina, catalog # 20020594). The libraries were barcoded by TruSeq® RNA Unique Dual Indexes (Illumina, catalog # 20022371) and quantified by qPCR on a Bio-Rad CFX Connect Real-Time PCR detection system with the NEBNext Library Quant Kit for Illumina (New England Biolabs, catalog # E7630L). The libraries were normalized to 10 nM, pooled and sequenced. RNA-seq. RNA sequencing was carried out at Johns Hopkins School of Medicine Genetic Resources Core Facility on a NovaSeq 6000 sequencing system (Illumina) with a S1 flow cell for 200 cycles, generating 100 bp paired-end reads.
GEO Accession
GSM3937566