title: GSM6206665: 9,000 cells of ADRCs 3 weeks after transduction with 6 factors; Mus musculus; RNA-Seq
library_construction_protocol: ADRCs were isolated from the inguinal and axillary fat pads of mice after sufficient anesthesia. The adipose tissues were washed with phosphate buffered saline (PBS), chopped fully with scissors, and then treated with 5 ml of collagenase type 1 (FUJIFILM, Cat# 037-17603) solution; subsequently, 2 mg/ml Hank's balanced salt solution was added after passing through a 0.22 μm mesh filter. Enzyme treatment was performed with gentle rocking at 37 ℃ for 30 to 45 min. The digested tissues were filtered through a 100 μm mesh filter and centrifuged at 500 × g for 5 min. The supernatant was removed and the pellet was stirred with 10 ml Dulbecco's modified Eagle medium (DMEM; Sigma Aldrich, Cat# D6046-500ML) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. The suspension was plated in 10 cm dishes (passage 0), and the medium was exchanged the day after isolating the ADRC. ADRCs were cultured under 5% CO2 at 37 ℃ conditions. The medium was changed every 2–3 days. After achieving the desired confluency of 80–90%, ADRCs were harvested by trypsinization for 5 min at 37 ℃  and pipetting to passage 1. After reaching approximately 80-90% confluency at passage 1, ADRCs were harvested and plated on 24 well plates at 30,000 cells/well. The next day, each viral solution was diluted with DMEM (with 10% FBS and 1% penicillin/streptomycin), and 10 μg/ml polybrene was added to the cells. The viral titer was confirmed using Lenti-XTM GoStixTM Plus (Cat #631280; Takara Bio), and the amount of each virus solution was adjusted to MOI=2. The mock cells were infected with a control lentivirus that did not contain the inserted target genes. The plates were incubated for 24 h. The medium was changed every 2–3 days. Transduced ADRCs were counted in 9,000 cells using CountessTM II FL (Invitrogen). As a negative control, 1,000 ADRCs without lentiviral induction were mixed with the sample. The Chromium Next GEM Single Cell 3' Reagent kit v3.1 (10X Genomics, Cat# PN-1000121) was used to label each cell and individual transcript with a unique molecular identifier (UMI), and cDNA libraries were generated according to the manufacturer's instructions.