GSM6914117: MCc_S1_L001_R1_001; Mus musculus; RNA-Seq
Nuclei preparation and FAC-sorting: Three virgin males, three virgin females and three mothers were sacrificed and brains were extracted in ice-cold oxygenated ACSF (containing (in mM): NaCl (124); NaHCO3 (26.2); glucose (11); KCl (2.5); CaCl2 (2.5); MgCl2 (1.3); and NaH2PO4 (1)). Each brain was then sectioned coronally at 1250 μm using a vibrating microtome (Campden Instruments). The lateral habenula was micro-dissected under a stereomicroscope, incubated for 5 min in 500 μl chilled 0.1X NP40 Lysis Buffer (Tris-HCL pH 7.4 10mM, NaCl 10mM, MgCL2 3mM, Tween-20 0.1%, NP40 0.1%, BSA 1%, DTT 1mM and RNase inhibitor), mechanically dissociated using a pellet pestle (15 goings and ongoings) and finally incubated on ice for 5 min. The suspension was then washed with 500 µL chilled wash buffer (Tris-HCL pH 7.4 10mM, NaCl 10mM, MgCL2 3mM, Tween-20 0.1%, BSA 1%, DTT 1mM, RNase inhibitor). Nuclei were then filtered through a 30-μm cell strainer, centrifuged (1000g, 10 minutes at 4°C) and resuspended in 500 µL of FACS buffer (Tris-HCL pH 7.4 10mM, NaCl 10mM, MgCL2 3mM, BSA 1%, DTT 1mM, RNase inhibitor). Positive GFP nuclei were sorted using a BD FACS Aria II flow cytometer (BD Biosciences) and collected in ice-cold FACS buffer. After sorting, nuclei were counted and immediately processed according to the 10X Chromium protocol. Briefly, an appropriate volume of each cell suspension containing 1000 cells from the three different conditions was combined with 10X Chromium reagent mix and samples were loaded into three separate lanes. Nuclei capture, lysis, mRNA reverse transcription, cDNA amplification and libraries were performed following 10X Genomics Chromium dual indexing Single Cell 3' V3.1 reagent kit instructions. Libraries were then multiplexed and sequenced according manufacture recommendations with paired-end reads using a HiSeq4000 platform (Illumina) with an expected depth of 250'000 reads per single nuclei. All the sequencing experiments were performed within the Genomics Core Facility of the University of Lausanne.