GSM4594396: FrozPit-FM3 [snRNA-seq]; Mus musculus; RNA-Seq
ii) Snap-frozen pituitaries were thawed on ice. For nuclei isolation, a modified protocol from 52 was followed, 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 with 10 strokes (loose pestle) in an individual autoclaved dounce glass homogenizer (1ml, VWR cat# 71000-514). The homogenate was filtered through a 40-micrometer cell strainer. The volume was measured and an equal volume of 50% OptiPrep was added. The gradient was centrifuged using an SW41 rotor at 9200 rpm and at 4C for 25 min. Nuclei were collected from the interphase between 30% and 35% OptiPrep. The nuclei were washed, resuspended either in 1x nuclei dilution buffer for snATACseq (10X Genomics) or in 1X PBS/0.04% BSA for snRNAseq, and counted on an Invitrogen Countess II cell counter. ii) Dissociated pituitary cells were cryopreserved in 90% FBS/10% DMSO and shipped to New York on dry-ice. Upon arrival, cells were stored in liquid nitrogen. On the day of an experiment, the vial was removed from liquid nitrogen on dry-ice, thawed quickly in a 37C waterbath, and cells were serially diluted with 10% FBS /DMEM until a minimum volume of 8ml was reached. Cells were centrifuged at 300xg for 5min, resuspended in 1ml of medium and counted. Nuclei were isolated from all cells remaining after the scGEM Drop-seq RNA-seq assay was done. Cells were centrifuged at 500g for 5min at 4C. The supernatant was carefully removed and 0.1X lysis buffer (1x: 10mM Tris-HCL pH 7.5, 10mM NaCl, 3mM MgCl2, nuclease-free H2O, 0.1% v/v NP-40, 0.1% v/v Tween-20, 0.01% v/v digitonin) was added. After 5min incubation on ice, 1ml of wash buffer was added. The nuclei were pelleted at 500xg for 5min at RT, resuspended either in 1X PBS/0.04% BSA for scGEM Drop-seq RNA-seq or in diluted nuclei buffer for snATAC-seq. Nuclei were counted and the concentration adjusted to run the given assay. snRNAseq were performed as described (10x Genomics, Pleasanton, CA; (24)), following the Single Cell 3' Reagents Kits V3 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 3' Chip kit A v2 PN-12036 or v3 chip kit B PN-2000060) 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-12062). 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
GSM4594396