title: GSM7668955: NSCs, M1; Rattus norvegicus; ssRNA-seq
library_name: GSM7668955
library_construction_protocol: NSCs were expanded as neutrospheres at 37°C, 5% CO2 for 3-4 days in proliferation culture medium, which contained DMEM/F12 supplemented with 1% glutamine (Gibco), 1% sodium pyruvate (Gibco), 1% NEAA (Gibco), 2% B27 (Gibco), 0.02 ng/mL EGF, 0.02 ng/mL bFGF, 1.83 ng/mL heparin and 1% streptomycin penicillin (Gibco). The proliferation medium was replaced every 2 days. Thereafter, the NSCs neutrospheres were gathered through centrifugation (300×g, 5min), and digested with 0.25% trypsin-EDTA (Gibco) at 37℃ for 18 min. After terminating digestion mixture using 10% FBS, the cells were collected by countrification and filtered through 70 μm and 40 μm mech of cell strainer successively to obtain the NSCs of single-cell suspension. 0.2% (W/V) of Col solution mixed with equal volume of NSCs single-cell suspension to obtain final 0.75, 1.5, 3 million/mL of cell packing density system. RNA-seq library preparation. Total RNA was isolated from each sample using the RNAmini kit (Qiagen, Germany). RNA quality was examined by gel electrophoresis and with Qubit (Thermo, Waltham, MA, USA). Strand-specific libraries were constructed using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), through reverse transcription using S1000TM Touch Thermal Cycler (Bio Rad) at 53°C (45 min) - 85°C (5 min) - 4°C cycle. The generated cDNA was amplified, and quality estimated using Agilent 4200 (performed by CapitalBio Technology, Beijing). The sequences of libraries were finally obtained using the Illumina Novaseq6000 sequencer with pair-end 150 bp (PE150).