title: GSM8209129: muscle, 30d, ScRNA-seq; Megalobrama amblycephala; RNA-Seq
library_construction_protocol: Dorsal skeletal muscle tissue was obtained from Megalobrama amblycephala. Place a sterile RNase-free culture dish containing an appropriate amount of calcium-free and magnesium-free 1 × PBS on ice, the tissue was transferred into the culture dish and cut it into 0.5 mm2 pieces, the tissues were washed with 1 × PBS, and remove as many non-purpose tissues as possible such as blood stains and fatty layers. Tissues were dissociated into single cells in dissociation solution in 37 ℃ water bath with shaking for 20 min at 100 rpm. Digestion was terminated with 1× PBS containing 10% fetal bovine serum , then pipetting 5-10 times with a Pasteur pipette. The resulting cell suspension was filtered by passing through 40μm stacked cell strainer and centrifuged at 300g for 5 min at 4 °C. The cell pellet was resuspended in 100ul 1× PBS (0.04% BSA) and added with 2-3 ml 1× red blood cell lysis buffer（Invitrogen, 00-4333） and incubated at room temperature for 2-4 min to lyse remaining red blood cells. After incubation, the suspension was centrifuged at 300g for 5 min at room temperature. Then the suspension was resuspended in 1× PBS (0.04% BSA) and centrifuged at 300 g for 3 min at 4 °C (repeat twice).The cell pellet was resuspended in appropriate volume of 1× PBS (0.04% BSA). Library was performed according to the manufacter's instructions (single cell 3'v3.1 protocol,10x Genomics). A Chromium Single Cell 3' Library & Gel Beads Kit v3.1 (10x Genomics) was used to generate oligo-dT-primed cDNA libraries according to the manufacturer's protocol. Subsequently, they were sequenced on an Illumina NovaSeq 6000 platform with a paired-end 150 read length.