GSM8881881: MC38-TMEM16F KO; Mus musculus; RNA-Seq
Homogenize Tissue: Remove any adherent tissue and homogenize the sample. Wash with PBS: Rinse the tissue with 5-10 ml of pre-cooled 1x PBS to remove blood stains. Weigh and Cut Tissue: Take approximately 0.05-0.1 g of tissue (about the size of a soybean) and cut it into small pieces that can fit into a centrifuge tube. Transfer to Tube: Place the small tissue pieces into a 1.5 ml centrifuge tube. Add Protectant: Add 1.3 ml of pre-cooled Metheni Tissue Protectant to ensure the tissue is fully saturated with the solution. Seal and Label: Seal the centrifuge tube with a sealing film and clearly mark the sample number on the tube. It's advisable to keep a backup sample. Storage and Shipping: Store the sealed centrifuge tube at 4°C and arrange for prompt shipment to the cold chain logistics company to maintain sample integrity. Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v3.1.Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNAs were then amplified by PCR to generate sufficient mass for library construction. Upon dissolution of the Gel Bead in a GEM, primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence were released and mixed with cell lysate and Master Mix. Barcoded, full-length cDNAs were then reverse-transcribed from poly-adenylated mRNA.