GSM9469382: Pooled sample of DEV16135 and DEV16171 12pcw Achilles tendons (whole); Homo sapiens; RNA-Seq
Nuclei isolation for fetal and adult tendon samples outlined in Table S1 was carried out following protocols for small tissues developed and published by Mimpen and colleagues from the Tendon Seed Network, found here: https://www.protocols.io/view/adaption-for-small-samples-nuclei-isolation-from-s-rm7vzyx75lx1/v1. Briefly, tendons were cut into ~1 mm pieces on dry ice and dissociated in CST buffer (292 mM NaCl, 20 mM Tris-HCL 7.5 pH, 2 mM CaCl2, 42 mM MgCl2, 0.5% CHAPS, 0.01% BSA, RNase and protease inhibitors) on a rotor for 2 min at 4°C. After addition of PBS with 2% BSA (bovine serum albumin) the suspensions were strained through 20 µm strainers (Greiner Bio-one). The nuclei-containing suspensions were then centrifuged at 500 g for 5 min at 4°C. Following centrifugation, the supernatant was discarded and the nuclei within the pellet were stained with DAPI and counted manually using a haemocytometer (NanoEntek DHC-N01) and fluorescence microscopy. Nuclear suspensions, diluted in PBS with 1% BSA to a concentration of 200-1000 nuclei/μl, were loaded onto a Chromium Next GEM Chip G (10x Genomics) with the aim of recovering 1,000-10,000 nuclei per sample. The samples were then processed using the Chromium Controller (10x Genomics) and prepared into libraries using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics) following the manufacturer's instructions. Libraries were indexed with the Single Index Kit T Set A (10x Genomics). Quality control assessments for cDNA and final libraries were conducted using D1000 or D5000 High Sensitivity ScreenTape (Agilent) assays on a 4150 TapeStation System (Agilent). The final libraries were pooled together and sequenced using a NovaSeq 6000 (Illumina) by Genewiz (UK) with a minimum sequencing depth of approximately 20,000 read pairs per expected nucleus.