GSM6755193: 290_D8; Mus musculus; RNA-Seq
At day 4, post cardiotoxin injections, whole hamstring muscle were sampled and homogenized in 4 ml of Trizol (Invitrogen) IKA T10 basic homogenizer on ice for 30 seconds for 3 times with 10 second intervals followed by incubation at RT for 30 minutes. Tissue debris were removed by centrifuging at 12,000xg and supernatants were mixed with 1/5 volume of chloroform. After spinning down at 12,000xg, 4°C for 15 mins, aqueous phases containing RNA were transferred to fresh tubes for RNA precipitation with isopropanol. The RNA pellets were further washed in 75% ethanol, air-dried and dissolved in RNAse/DNase free water. RNA-Seq libraries were generated in using the Illumina Stranded mRNA Library Prep Ligation kit (illumina, 20040534) and IDT for Illumina RNA UD Indexes (illumina, 20040553) according to the standard manufacturer's protocol (illumina, document # 1000000124518 v02, April 2021) described briefly as follows. 80 ng of total RNA was enriched for mRNA using an oligo-dT bead isolation method. The purified mRNA was then subjected to a heat fragmentation step and copied into first strand complimentary DNA (cDNA) using reverse transcriptase and random primers. The resulting cDNA was converted into dsDNA in the presence of dUTP to prevent subsequent amplification of the second strand and thus maintaining the strand orientation of the original mRNA. The 3' ends of the cDNA were adenylated and pre-index anchors were ligated. The libraries were then amplified with 13 cycles of PCR incorporating unique indexes for each sample to produce libraries ready for sequencing. The libraries were quantified on the Perkin Elmer LabChip GX Touch with the DNA High Sensitivity Reagent kit (Perkin Elmer, CLS760672). Libraries were pooled in equimolar ratios.