GSM9822023: Day 28 G2 clone 1; Homo sapiens; RNA-Seq
Cells were fixed with glyoxal as described to preserve RNA integrity during sorting (39). Following fixation cells were saponin-permeabilized and using FACS were sorted into G1, S, and G2 populations. Cells were first gated based on DAPI content, followed by refinement using mKO2-CDT1 and Clover-GMNN marker expression, and were collected directly into TRI Reagent for RNA extraction. Total RNA was extracted using TRI Reagent (Sigma, T9424) followed by organic phase separation and isopropanol precipitation. RNA purity was assessed by spectrophotometry. Poly(A)+ RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S). Strand-specific libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7760S). Libraries were amplified for 10–18 PCR cycles based on test amplifications carried out on a small portion of the sample. Following AMPure XP bead purification, libraries were quality assessed by Agilent Bioanalyzer, quantified by KAPA Library Quantification Kit. Sequencing was performed (75 bp single-end) to an average depth of ~24 million reads per sample.