title: GSM7918210: Female Skeletal Muscle anti-PD-1 T-Cells 5; Mus musculus; RNA-Seq
library_construction_protocol: Hearts were perfused with 3-4 mL of HBSS prior to digest. Diaphragm and lower limb soleus muscles were pooled together for skeletal muscle samples. Tissue from two mice were pooled together for digestion. Digestion took place at 37C for 1 hr in HBSS supplemented with 300U/mL collagenase, 5U dispase, and 10μg/ ml DNaseI.  Every 15 minutes samples were triturated 10 times using a 10mL pipette. Following digestion, samples were passed through a 70μm nytex nylon mesh and flooded with calcium and magnesium free HBSS supplemented with 0.5% FBS and 2mM EDTA. Red blood cell lysis was performed using Gey's buffer. PI- CD45.1+ CD11b- CD11c- B220- CD90.2+ TCRβ+ cells were sorted directly into FBS followed by two washes with Dulbecco's PBS w/calcium & magnesium. Sorted cells were flash frozen in RLT Buffer supplemented with 2-Mercaptoethanol (10μl/mL) and stored at -800 C until RNA extraction. After thaw, samples were homogonized using a 20-gauge needle. RNA was isolated using the RNeasy Micro kit and the Rneasy Mini kit with the optional Dnase digest step. RNA concentration and quality were assessed using the RNA 6000 Pico Assay. Further pooling of extracted RNA was performed to bring skeletal muscle samples to the necessary minimum input for library preparation with each final sample containing RNA from 2-12 total starting mice. Each pooling performed for skeletal muscle samples was also used for corresponding heart samples. KAPA mRNA HyperPrep Kit