GSM7814394: WT 3-2 e; Homo sapiens; RNA-Seq
Individual chondronoids were snap frozen and pulverized. Each of the three independent differentiations was considered a biological replicate. Chondronoids from the same differentiation are indicated as such (-1,-2 etc.). The pulverized tissue was resuspended in TRIzol, and extracted by chloroform according to manufacturer's protocol. The aqueous phase was collected and RNA was purified using Direct-zol RNA Microprep kit (Zymo Research) according to manufacturer's protocol. High throughput three prime digital gene expression (HT-3'DGE) was adapted from the Soumillon et al. protocol (Soumillon et al. BioRxiv https://www.biorxiv.org/content/10.1101/003236v1) to perform reverse-transcription reactions using a dedicated RT primer containing sample and unique molecular identifier (UMI) sequences, to generate four subpools of 24 samples each. After second strand synthesis, Illumina adapters were added using the Nextera Flex tagmentation protocol substituting the normal P500 primer with an ad-hoc primer. Final libraries were cleaned using SPRI beads at 0.7X and quantified using the Fragment Analyzer and qPCR before being loaded for paired-end sequencing using the Illumina NextSeq500 in high-output paired-end mode (20/57 nt reads).