title: GSM8853858: Adult, soleus, Adult 7, RNAseq; Mus musculus; RNA-Seq
library_construction_protocol: At E18.5 pregnant females were euthanized to acquire embryos. Adult mice were exposed to CO2 at a constant rate for 5 minutes and secondary euthanasia was performed via cervical dislocation by a trained investigator. E18.5 embryos were collected from pregnant females, euthanized via decapitation, and anatomically sexed. To minimize the signal of X-chromosome silencing factors (e.g., Xist, Tsix), only muscle samples from male mice were used for ATAC-seq and RNA-seq analysis. Four hindlimb skeletal muscles were chosen for dissection based on previously described differences in slow myofiber content. The m. soleus and m. vastus intermedius represent slow-biased muscles, and the m. gastrocnemius and m. vastus lateralis represent fast-biased muscles. Muscle samples were collected from adult male (N = 6) and E18.5 (N = 6) mice and placed in TRIzol reagent (Invitrogen). Muscle samples were pulverized using an electric homogenizer and subjected to a phenol-chloroform reaction. Total RNA extraction and purification was performed using a Zymo Direct-zol RNA Microprep Kit following the manufacturer's instructions. RNA was eluted using nuclease-free water and assessed for quantity and quality. Samples were quantified using a Qubit and nanoDrop to determine their 260/230 and 260/280 values. Aliquots of samples were run on a TapeStation to determine their RNA integrity number (RIN) scores. Only samples with a RIN score above 7.0 were retained for library preparation and processed for sequencing. RNA-seq libraries were prepared by the Harvard Bauer Core Facility using the Kapa mRNA HyperPrep Kit and automated using a PerkinElmer Sciclone NGS Workstation. RNA was reverse transcribed into cDNA using random primers. Total RNA was quantified using a fluorimetric Quant-iT RiboGreen RNA Assay Kit and total RNA integrity was assessed using an Agilent BioAnalyzer 2100 RNA Nano Kit. All libraries were pooled and assessed for quality using qPCR. To obtain a minimum of 30 million reads per sample, all deep sequencing was performed using 2 x 100-bp PE reads on an Illumina NovaSeq6000 instrument. Demultiplexed sequencing results were analyzed following ENCODE RNA-seq guidelines (www.encodeproject.org/about/experiment-guidelines/).