GSM5391771: Sample 3-R: Pit_Aged_HF3_fragm_snRNA; Homo sapiens; RNA-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 PBS/0.04% BSA for snRNAseq (10X Genomics). snRNAseq were performed as described (10x Genomics, Pleasanton, CA; (24)), following the Chromium Next GEM Single Cell 3' Reagents Kits V3.1 User Guidelines. Nuclei were filtered and counted on a Countess instrument. The amount of nuclei loaded on the chip was determined based on the 10X Genomics protocol. The 10X chip (Chromium Single Cell Chip kit G PN-2000177) was loaded to target a minimum of 1,000 nuclei final. Reverse-transcription was performed in the emulsion and cDNA was amplified before library construction following the Chromium protocols v2 or v3. Each library was tagged with a different index for multiplexing (Chromium i7 Multiplex kit PN-220103). Quality control and quantification of the amplified cDNA 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
GSM5391771