title: GSM7901467: Uninjured-Lane 1-1; Mus musculus; RNA-Seq
library_name: GSM7901467
library_construction_protocol: Tissue samples were collected from n=4 uninjured mice and n=4 mice at 28d after crush SCI. Mice were perfused with ice cold heparinized saline that was prepared using RNAse- and DNAse- free water and 10X PBS for 2 minutes at 7ml/min for blood clearance. Spinal cords were rapidly dissected on ice chilled blocks. For SCI samples, the lesion core center was identified and a tissue block of 1 mm rostral and caudal was then rapidly removed. Anatomically equivalent regions of spinal cord were taken from uninjured mice. Tissue samples were rapidly snap frozen in microcentrifuge tubes maintained in a dry ice bath and stored at -80°C until further processing. To avoid potential batch effects, frozen tissue samples were all processed at the same time. To enrich for astrocyte nuclei, we used Sox9 antibody-binding and magnet-assisted nuclear immunoprecipitation (MAN-IP) using well characterized procedures{Krishnaswami, 2016 #5364;Bhattacharyya, 2019 #5615;Batiuk, 2017 #5624} as follows. Tissue samples were first gently dissociated by trituration and pelleted by centrifugation. Nuclei were extracted from cell pellets by gentle resuspension in ice-cold lysis buffer (10mM Tris buffer, 10mM NaCl, 3 mM MgCl2, 0.1% Nonidet P40 Substitute). Nuclei were pelleted by centrifugation (500RPM (Model 5415R, Eppendorf) for 5 min at 4°C) and then resuspended in Nuclei Wash and Resuspension Buffer (NWRB) (1xPBS, 1% BSA, 0.2U/uL RNAse inhibitor) before being washed once more in NWRB and then filtered using a 5 mL Polystyrene Round-Bottom Tube with 35 µm Cell-Strainer Cap and concentrated to a nuclei concentration of 1000 nuclei/µL (1 x 106 nuclei/mL), resuspended and incubated with Sox9 rabbit monoclonal antibody (ThermoFisher, Cat#72016) for 30 minute and then centrifuged at 700g for 10 minutes. Pellets were resuspended in 80μl of MACS Buffer composed of 1X PBS (Tissue Culture grade; Ca2+, Mg2+ free), 0.5% Nuclease free Bovine Serum Albumin (BSA), and 2mM EDTA, and then incubated with anti-Rabbit IgG Microbeads (Miltenyi, Cat# 130-048-602) for 20 minutes at 4°C. After washing with 1ml of MACS buffer at 300g for 10 minutes at 4 ̊C, immunolabeled nuclei were enriched by magnetic separation using MACS MS columns (Miltenyi Cat# 30-042-201, 130-042-102 and 130-042-303). Pellets were resuspended in an appropriate volume of Resuspension Buffer to achieve a final concentration of 1000 nuclei/µL (1 x 106 nuclei/mL) and immediately processed for Library preparation using Chromium Next GEM Single Cell 3' v3.1 kits using manufacturer's instructions. Eight library samples were then processed simultaneously for RNA-Sequencing on 2 lanes (Lane 1: injured-1, injured-2, uninjured-1, uninjured-2; Lane 2: injured-3, injured-4, uninjured-3; uninjured-4) of Illumina NovaSeq S4 with at least 300M reads per library sample, conducted in house by the UCLA Technology Center for Genomics & Bioinformatics using pair end reads (2x100). The library preparation and sequencing were performed following 10X Genomics Chromium protocols.