title: GSM8853769: Adult, vastus intermedius, Adult 1, ATACseq; Mus musculus; ATAC-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 = 3) and E18.5 (N = 3 pooled litters) mice. To ensure at least 50,000 cells per muscle for ATAC-seq analysis, each E18.5 biological replicate included pooled muscles (both left and right sides) from up to 5 male embryos belonging to the same litter. Each sample was subjected to a protocol that isolates muscle nuclei from undigested tissue (Young et al., 2022). Cell counting was performed using trypan blue and a hemocytometer under an inverted microscope. Only samples having a death rate below 25% were subjected to an optimized ATAC-seq protocol (Corces et al., 2017). Briefly, muscle nuclei were lysed to extract DNA and treated with Tn5 transposase to tag transcriptionally accessible fragments of DNA not bound to histones (i.e., potential enhancers). Transposed DNA was purified using a Zymo DNA Clean and Concentrator Kit following the manufacturer's instructions. Samples were then primer barcoded and PCR-amplified to generate ATAC-seq libraries. Libraries were treated for fragment size selection using the OMEGA Bead Purification Protocol and concentrations were determined using the KAPA Library Quantification Complete Kit. ATAC-seq library quality was assessed by the Harvard Bauer Core Facility using the Agilent Bioanalyzer 2100 DNA High Sensitivity Kit and the Agilent TapeStation 4200 (D1000 ScreenTape assay). All libraries were pooled and assessed for quality using qPCR and Illumina MiSeq Nano. Library pools were adjusted based on the Illumina MiSeq Nano results to optimize the distribution and final loading concentration. To obtain a minimum of 50 million reads per sample, all deep sequencing was performed using 2 x 100-bp PE reads on an Illumina NovaSeq 6000 instrument. Demultiplexed sequencing results were analyzed following ENCODE ATAC-seq guidelines (www.encodeproject.org/about/experiment-guidelines/).