GSM5391763: Sample 2-A: Pit_Adult_HF2_fragm_snATAC; Homo sapiens; ATAC-seq
Briefly, and all on ice, RNAse inhibitor (NEB MO314L) was added to the homogenization buffer (0.32 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 5mM CaCl2, 3mM Mg(Ac)2, 0.1% IGEPAL CA-630), 50% OptiPrep (Stock is 60% Media from Sigma; cat# D1556), 35% OptiPrep and 30% OptiPrep right before isolation. Each pituitary was homogenized in a dounce glass homogenizer (1ml, VWR cat# 71000-514), and the homogenate filtered through a 40 mm cell strainer. An equal volume of 50% OptiPrep was added, and the gradient centrifuged (SW41 rotor at 9200rpm; 4C; 25min). Nuclei were collected from the interphase, washed, resuspended in 1X nuclei dilution buffer for snATACseq (10X Genomics). snATACseq was performed as described (10x Genomics, Pleasanton, CA), following the Chromium Next GEM Single Cell ATAC Reagent Kits V1.1 User Guide. Nuclei were counted on a Cellometer instrument, and the final concentration was used to calculate the volume of nuclei stock to use. Transposition was performed in 10 microliter at 37C for 60min. Then the Chromium Chip H (PN-2000180) was loaded to target 1,000-10,000 nuclei. Barcoding was performed in the emulsion for 12 cycles following the Chromium protocol. Each library was tagged with a different index for multiplexing (Chromium i7 Sample Index N, Set A kit PN-3000262). Quality control and quantification of the libraries were assessed on a Bioanalyzer (High-Sensitivity DNA Bioanalyzer kit). Sequencing was carried out at the NYGC on Illumina Novaseq using 98+26 paired-end reads.
GEO Accession
GSM5391763