Ovomucoid

ATAC-seq processing followed the original protocol (Buenrostro et al., 2013 (link)), which involves enzymatic dissociation of tissue using papain inactivated by ovomucoid (Worthington) followed by aliquoting of 50,000 cells, nuclear isolation using an IGEPAL solution, transposition using Nextera Tn5 transposase (Illumina), barcoding and amplification (8-11 cycles), quality control via gel electrophoresis, quantification via qPCR with DNA Standards (Kapa Biosystems), and massively parallel 50 bp paired end (PE) sequencing on an Illumina HiSeq2000 or HiSeq2500 to acquire ∼25M fragments per sample. For each donor, GZ and CP samples were dissected, library prepped, and sequenced within the same batch to prevent batch effects correlated with the biological condition of interest. Following preparation of a homogeneous suspension of cells isolated from the GZ or CP, 3-4 technical replicates consisting of independent nuclear isolations, Tn5 tagmentation, and library preparation were generated. phNPC samples were processed for ATAC-seq library preparation at 0 wks (3 replicates - independent nuclear isolations, Tn5 tagmentation, and library preparation from the same differentiation), 2 wks (3 replicates), 4 wks (2 replicates), and 8 wks (3 replicates) post differentiation as above.

Two and four days after transfection, cultured OPCs were rinsed twice with Hanks' balanced salt solution without calcium and magnesium (Thermo Fisher Scientific), and then trypsinized with 0.25% trypsin (Thermo Fisher Scientific) in the incubator for 5 min. Cells were resuspended by pipetting and trypsinization was stopped by adding an equal volume of DPBS without calcium and magnesium (Thermo Fisher Scientific) containing 0.25% ovomucoid (Worthington) and 0.25% bovine serum albumin (Sigma-Aldrich). After centrifugation at 300 × g for 5 min, the cell pellet was resuspended in 0.4 ml of FACS buffer (DPBS without calcium and magnesium containing 1 × B-27, 1 × N-2, 2 mM EDTA, 5.56 mM glucose, and 25 mM HEPES-Na pH 7.0) and kept on ice. Cell suspension was filtered through a 40 μm cell strainer and EGFP+ cells (MFI cut-off 103) were sorted using the FACSAria II (BD Biosciences). EGFP+ cells were collected into 300 μl FACS buffer at 4°C for RNA isolation, or in 300 μl proliferation medium containing 25 mM HEPES-Na pH 7.0 at room temperature for re-culture experiments. For RNA isolation, sorted cell suspension was centrifuged for 8 minutes at 300 × g at 4°C and the cell pellet lysed in 350 μl RLT buffer with 1% 2-mercaptoethalnol (Sigma). For re-culture experiments, sorted cells were re-plated into proliferation media and allowed to recover for two days before fibrinogen treatment.