GSM5425887: CLP+MLT4_heart; Mus musculus; RNA-Seq
Samples were collected and flash-frozen in liquid nitrogen and, then treated by Trizol in -80℃ until RNA extraction. Extracting total RNA from tissues by Trizol. 1.5% agarose gels were used to monitor the RNA degradation and contamination. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The next RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 3.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Library preparation for RNA sequencing. A total amount of 1μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5X).First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEB Next Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments with right length, the library fragments were purified with AMPure XP system(Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with sizeselected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 System.