GSM9009205: Myelin Spheres Week 8; Homo sapiens; RNA-Seq
Week 8 or 15 old NMs and MyS (4-6 spheroids per sample) were transferred to 250 µL of ice-cold homogenization buffer (HB; 10 mM Tris pH 7.8, 0.1 mM EDTA, 320 mM sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 0.1% NP40, 1X complete protease inhibitor (Roche), 1 mM β-mercaptoethanol, and RNase inhibitor (Promega)) for 3 min to preserve nuclear integrity. Samples were gently homogenized using pestle A (5 strokes) followed by pestle B (15 strokes) and the homogenates were passed through 70 µm EASYstrainer (Greiner). The homogenizer and the strainer were washed with additional 270 µL of HB, and 520 µL of gradient medium (10 mM Tris pH 7.8, 5 mM CaCl2, 50% Optiprep, 3 mM magnesium acetate, 1X complete protease inhibitor, 1 mM β-mercaptoethanol, and RNase inhibitor) was added to the samples. This solution was carefully layered onto a cushion solution (29% Optiprep, 150 mM KCl, 30 mM MgCl2, 60 mM Tris pH 7.8, and 250 mM sucrose) and centrifuged at 9000 rpm for 25 min at 4 °C. The supernatant was discarded carefully, and nuclei pellets were then resuspended in 25 µL of resuspension buffer (1X PBS, 2% bovine serum albumin (BSA)), filtered through Flowmi 40 µm cell strainers, and counted using a LUNA automated cell counter (Logos Biosystems) following the manufacturer's instructions. The diluted nuclei suspensions were subsequently loaded onto the 10X Chromium Single Cell Platform (10X Genomics) (Next GEM Single Cell 3′ library and Gel Bead Kit v3.1) in accordance with the manufacturer's protocol (10X User Guide; CG000315, Revision E). Steps including generation of gel beads in emulsion (GEMs), barcoding, GEM-RT cleanup, complementary DNA amplification, and library construction were all performed according to protocol. Quality of individual samples was checked with a Fragment Analyzer (Agilent), and library quantification was done using a Qubit 4.0 (ThermoFisher Scientific) before pooling the libraries. The final pooled library was sequenced on a NovaSeq6000 (Illumina) instrument at the VIB Nucleomics Core, using the Illumina NovaSeq 6000 v1.5 sequencing kit S4 200 paired-end reads (28-10-10-90), 1% PhiX with 100-base-pair paired-end reads.