title: GSM9107834: AT SCM 3; Mus musculus; RNA-Seq
library_construction_protocol: For FACS sorted SCM muscle, SCM muscles were manually dissected from surrounding tissue, manually dissociated and then digested for 1 hour at 37C in 2U/mL Collagenase B (Roche, #11088831001), Dispase II (Roche, # 04942078001), 50 mM HEPES/KOH pH 7.4, 150 mM NaCl. Cells were then resuspended in 2x volume 10% FBS  in PBS to inactivate digestion enzymes. Samples were filtered through 70 uM filter, spun and supernatant spun again. Then filtered through a 40 uM filter to yield a single cell suspension. Samples were spun down and resuspended in 5% FBS PBS prior to sorting on a FACS Aria for tdTomato+ cell populations with DAPI as a live/dead marker. Data were analyzed with FACSDiva and FlowJo software. Following FACS sorting RNA was isolated from tdTomato+ cells with miRNeasy microelute kit (Qiagen) per manufacturer's instructions. For FACS sorted samples low input RNA unstranded sequencing was used. One nanogram of total RNA was used to create SPIA-cDNA with a Tecan Ocation RNA seuqncing stem v2 protocol (Tecan, #7102-32) per manufacturer's instructions. 500 ng of purified cDNA was sheered ina Covaris LE220 focused ultra sonicator using a 96 microtube-50 AFA fiberr plate and target base pair size of 300 bp. Following sheering libraries were created using KAPA Hyper-Prep kit (Roche, #KK8504) per manufacturer's instructions based on initial cDNA input. UDI DNA indexes were used from Illumina (20022370).