title: GSM6865282: Differentiation day6; Homo sapiens; RNA-Seq
library_construction_protocol: Total amounts and integrity of RNA were assessed using the RNA Nano 6000  Assay Kit. total RNA was used for the construction of sequencing libraries. During the differentiation of cardiac organoids, the cells were harvested on days 0, 2, 3, 4, 6, and 30, and 10 organoids were harvested at each time point.Total amounts and integrity of RNA were assessed using the RNA Nano 6000  Assay Kit of the Bioanalyzer 2100 system.Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in  First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using  random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to  degrade the RNA. Second strand cDNA synthesis was subsequently performed using  DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends  via exonuclease/polymerase activities. After adenylation of 3' ends of DNA  fragments, Adaptor with hairpin loop structure were ligated to prepare for  hybridization. In order to select cDNA fragments of preferentially 370~420 bp in  length, the library fragments were purified with AMPure XP system (Beckman  Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by  AMPure XP beads, and the library was finally obtained.After  the construction of the library, the library was initially quantified by Qubit2.0  Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by  Agilent 2100 bioanalyzer.After insert size meets the expectation, qRT-PCR is used to  accurately quantify the effective concentration of the library to ensure the quality of the  library