GSM9825215: VTA_smoke; Mus musculus; RNA-Seq
brain tissues were finely minced into ~0.1 cm fragments and transferred into a mixture of 1 ml lysis buffer and 1 ml PBS. Samples were washed twice in PBS by centrifugation to remove the supernatant. Tissues were then incubated with 2 ml of pre-chilled lysis buffer on ice for 2 minutes for pre-lysis. Homogenization was carried out using a Dounce homogenizer (Sigma), followed by gentle pipetting (5–6 times) to ensure complete dissociation. The homogenate was kept on ice for 6 minutes, after which 2 ml of pre-chilled 4% BSA was added to dilute the mixture and terminate lysis. This was followed by pipetting (6–8 times) with a Pasteur pipette. Samples were centrifuged at 300 × g for 5 minutes at 4°C, and the supernatant was discarded. The resulting pellet was resuspended in 2 ml of lysis buffer containing 4% BSA and incubated on ice for an additional 3 minutes. Cellular debris was removed using the Miltenyi Biotec Debris Removal Solution (Order no. 130-109-398). After a second centrifugation under the same conditions, the nuclei were resuspended in a nuclear resuspension buffer (1× PBS, 2% BSA, 0.1% RNase inhibitor, Roche). Nuclei were then filtered through a 20-μm cell strainer and counted. Final libraries were sequenced using an Illumina NovaSeq 6000 platform with paired-end 150 bp reads by LC-Bio Technology Co. (Hangzhou, China).