GSM4314833: H9_P3CNCC_RNAseq_replicate4; Homo sapiens; RNA-Seq
Total RNA was extracted from a 6-well of hESC, early (P1 and 2) and late (P3 and P4) hCNCCs and day 9 chondrocytes differentiated from hCNCCs using Trizol reagent (Invitrogen) for four independent differentiations. 10μg RNA was purified twice by Dyna1 oligo(dT) beads (Invitrogen) to enrich for poly(A)+ mRNA. The mRNA was then fragmented using 10X fragmentation buffer (Ambion) for exactly 5 min and purified by ethanol precipitation with sodium acetate and RNase-free glycogen. First strand synthesis was performed using Random Hexamer Primers (Invitrogen) and SuperScript II (Invitrogen), followed by second strand synthesis using DNA PolI and RNaseH (Invitrogen), and cDNA was purified using Nucleospin Gel and PCR Cleanup (Takara). All of the cDNA was used for library preparation by end repair, A-tailing and adapter ligation (NEB). The samples were treated with USER enzyme, purified using XP SPRI beads then subjected to dual size selection using XP SPRI beads using bead ratios 0.55x to remove >700bp followed by 0.85x to recover >200bp sized cDNA. Size-selected cDNA was amplified using NEBNext HiFi 2X PCR mix and Dual Index Primers (NEB, E7600) for 7-10 cycles (as determined by qPCR). Libraries were then purified using XP SPRI beads, and quantified using Qubit dsDNA HS assay kit and pooled for sequencing using average library size (bp) from Bioanalyzer and concentration from KAPA quantification (Kapa Biosystems). Libraries were sequenced using NextSeq 500 (2x 75 bp).
GEO Accession
GSM4314833