GSM4264271: Skeletal muscle Lane 1; Mus musculus; RNA-Seq
A collagenase digestion was performed on the flexor digitorum brevis muscle in a dish containing DMEM with 1% penicillin/streptomycin, 1% fetal bovine serum, and 2mg/mL Collagenase Type II, kept at 37C for 1.5 hours. After 1.5 hours the muscle was transfered to a non-collagenase media and titurated. Single myocytes were isolated using a large particle fluorescent activated cell sorter using a flow channel size of 500 um. The COPAS SELECT Flow Pilot Platform was used to isolate cell populations. These sorted cells were placed individually into 96-well plates. Capture plate wells contained 5 µl of capture solution (1:500 Phusion High-Fidelity Reaction Buffer, New England Biolabs; 1:250 RnaseOUT Ribonuclease Inhibitor, Invitrogen). Single cell libraries were then prepared using the mcSCRB-seq protocol. Briefly, cells were subjected to proteinase K treatment followed by RNA desiccation to reduce the reaction volume. RNA was subsequently reverse transcribed using a custom template-switching primer as well as a barcoded adapter primer. The customized mcSCRB-seq barcode primers contain a unique 6 base pair cell-specific barcode as well as a 10 base pair unique molecular identifier (UMI). Transcribed products were pooled and concentrated, with unincorporated barcode primers subsequently digested using Exonuclease I treatment. cDNA was PCR-amplified using Terra PCR Direct Polymerase (Takara Bio). Final libraries were prepared using 1ng of cDNA per library with the Nextera XT kit (Illumina) using a custom P5 primer.
GEO Accession
GSM4264271