GSM8641972: Sorted nuclei, muscle; Homo sapiens; RNA-Seq
Skeletal muscle samples were obtained from the vastus lateralis muscle under local anesthesia and issected into visible skeletal muscle and adipose tissue components, and each component was immediately placed in a tube and prefilled with at least 5 ml of Allprotect® Tissue Reagent. Preserved skeletal muscle specimens stored in ATR were thawed on ice. Ice cold PBS was added and pipette-mixed with ATR to dilute and remove supernatant. This process was repeated until all ATR was removed. Tissue was subsequently rinsed twice in ice cold PBS to remove any remaining ATR, weighed, and resuspended in pBSA (PBS + 0.04% BSA) + 1U/mL RNaseOut (RNaseOUT™ Recombinant RNase Inhibitor, Thermo Fisher Scientific, Waltham, MA, catalogue no. 10777019). Manual tissue dissociation was performed with a disposable micro-tissue homogenizer. Cell concentrations were estimated with a hemocytometer. Single cell suspensions were strained sequentially through 70 µm and 40 µm filters, with washes between each step. For sorted samples, cells were stained with a final concentration of 25 µg/mL anti-Nuclear Pore Complex conjugated to Alexa Fluor® 594 (NPC-AF594) antibody (BioLegend, San Diego, CA, catalogue no. 682202) and NPC+ cells isolated on a Becton Dickinson FACS ARIA Fusion using a 70 µm nozzle. For nuclei isolation, cells were incubated with Nuclear Extraction Buffer (Miltenyi Biotec, Gaithersburg, MD, catalogue no. 130-128-024) containing 1 U/mL RNaseOut at 4˚C for 5 minutes. Following the incubation, suspensions were centrifuged at 300 x g, 4°C, 5 minutes, supernatant removed, and nuclei resuspended in pBSA. Samples were sorted on ice into pBSA before downstream processing. Note that “sorted nuclei” refers to cells that were sorted prior to nuclei extraction. Libraries were prepared using a Chromium Single Cell 5' Library & Gel Bead Kit v2 (P/N 1000263, 10x Genomics). GEM-RT was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad; P/N 1851197). Following retrotranscription, GEMs were broken and the single-strand cDNA was purified with DynaBeads MyOne Silane Beads. cDNA was amplified using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module:. Amplified cDNA product was purified with the SPRIselect Reagent Kit (0.6× SPRI). Indexed sequencing libraries were constructed using the reagents in the Chromium Single Library construction kit, following these steps: (1) end repair and A-tailing; (2) adaptor ligation; (3) post-fragmentation, end repair, and A-tailing double size selection cleanup with SPRI-select beads; (4) sample index PCR and cleanup. The barcoded sequencing libraries were diluted at 3 nM and sequenced on Illumina NovaSeqX.