Bulk RNA sequencing of chemical-induced intermediate cells

This study aims to investigate the individual effect of cocktail VCRTc on the direct reprogramming process.We collected cells as follows: mouse chondrocytes (mchons), mouse embryonic fibroblasts(MEFs), MEFs treated by a chemical cocktail VCRTc for 3 days (3d-VCRTc) and 6 days (6d-VCRTc), and MEFs treated by individual chemical V, C, R, T, ce for 6 days, respectively. Then we did RNA extraction,reverse transcription and library preperation before bulk RNA seq. We uncovered that MEFs treated with chemicals have an upregulation of chondrocytes enriched genes; and a downregulation of MEF enriched genes,indicating that chemical treatment drove MEF toword chondrogenic lineage before the 2nd stage of differentiation. RNA-seq was modified from a previous method. Briefly, RNA was extracted from samples by Trizol reagent (TAKARA), reverse transcription was conducted by SuperScript II reverse transcriptase (Invitrogen), double strand cDNA was conducted using NEBNext mRNA second strand synthesis kit (NEB), double strand DNA was cleaned with AMPure XP beads (Beckman Coulter). 3’end enriched sequencing library was constructed with Nextera XT kit (Illumina) and sequenced on Illumina X-Ten platform. There were 3 samples for each group and 5 replicates of sequencing library for each sample.