GSM8712448: Atrial tissue WT7; Mus musculus; RNA-Seq
The atria were lysed with 250 μL TRI reagent (T9424, Sigma-Aldrich, Inc., St. Louis, MO) per atrial pair. The lysates were partially homogenized by brief mechanical homogenization at 4 °C and incubated for 5 minutes at room temperature. Total RNA was then isolated from the supernatant using the Direct-zol RNA Miniprep Kit (R2050, Zymo Research, Irvine, CA) according to the manufacturer's protocol. 200 μL fresh TRI reagent was added to each leftover unhomogenized tissue sample, and the samples were completely homogenized by additional mechanical homogenization at 4 °C. Total RNA was extracted from these homogenates upon addition of chloroform according to the manufacturer's protocol. The Direct-zol kit-extracted and Tri Reagent/chloroform-extracted RNA fractions from each sample were combined for RNA-seq. Total RNA sample quantity, purity, and integrity were determined by spectrophotometric absorbance at 260 nm, the 260/280 nm absorbance ratio, and gel electrophoresis, respectively. The total-RNA samples were shipped to GENEWIZ (Azenta, South Plainfield, NJ) on dry ice for standard RNA-seq at a targeted depth of 30 million reads per sample. The yield for 9 of the 10 whole-atrium samples exceeded 30 million reads per sample; one of the atrium samples had a yield of 24.3 million reads. At Azenta, RNA sample quantity was confirmed using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA), and RNA integrity was confirmed using the Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA). Library preparation with stranded polyA selection was then performed. The libraries were sequenced using a 2x150bp Paired End (PE) configuration on the Illumina® NovaSeq™ 6000 platform. Raw sequence data (.bcl files) generated from Illumina platform were converted into .fastq files, de-multiplexed using Illumina's bcl2fastq 2.17 software, and delivered by File Transfer Protocol (FTP).