title: GSM7349674: P13_pseudarthrosis_periosteum; Homo sapiens; RNA-Seq
library_construction_protocol: Nuclei extraction was performed on snap-frozen human periosteum samples from PA site and IC of patient P13 and from PA site of patient P5. RNA integrity was checked beforehand on identical tissue samples (RIN > 6.5). Periosteum samples were briefly defrosted in nuclei extraction buffer before mincing and nuclei extraction. Collected tissues were minced and placed 5 min in ice-cold Nuclei Buffer (NUC101, Merck) before mechanical nuclei extraction using a glass douncer. Extraction was performed by 20 strokes of pestle A followed by 10-20 of pestle B. Nuclei suspension was filtered, centrifuged and resuspensed in RNAse-free PBS with 2% Bovine Serum Albumin and 0.2 U/µL RNAse inhibitor. A second step of centrifugation was performed to reduce contamination by cytoplasmic RNAs. Sytox™ AADvanced™ was added (1/200) to label nuclei and Sytox-AAD+ nuclei were sorted using Sony SH800. The snRNA-seq libraries were generated using Chromium Single Cell Next GEM 3′ Library & Gel Bead Kit v.3.1 (10x Genomics) according to the manufacturer's protocol. Briefly, 10 000 to 20 000 nuclei were loaded in the 10x Chromium Controller to generate single-nuclei gel- beads in emulsion. After reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR. Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq (Illumina) with 28 cycles of read 1, 8 cycles of i7 index and 91 cycles of read 2. Sequencing data were processed using the Cell Ranger Count pipeline and reads were mapped on the HG38 2020-A reference human genome, with intronic and exonic sequences.