GSM7845344: MT1_D7_rep2; Homo sapiens; RNA-Seq
Total RNA was isolated from cells at the various stages of osteogenic differentiation using TRIZOL reagent (sigma). RNA sequencing library was prepared using Illumina-compatible NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England BioLabs) following manufacturer's protocol. 500 ng of total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified using JetSeq magnetic beads and was end-repaired, adenylated and ligated to Illumina adapters as per NEBNext® Ultra™ II Directional RNA Library Prep protocol followed by second strand excision. Adapter ligated cDNA was purified using JetSeq Beads and was subjected to 11 cycles for Indexing and to enrich the adapter-ligated fragments. Final PCR products (sequencing library) were purified with JetSeq Beads, followed by library quality control check. Illumina-compatible sequencing libraries were quantified by Qubit fluorometer and fragment size distribution was analyzed on Agilent 2200 TapeStation. The Illumina-compatible sequencing libraries showed an average fragment length of 496 bp and sufficient concentration to obtain the desired amount of sequencing data. The libraries were paired-end sequenced on Illumina Sequencer