title: GSM5393145: MSC_P6_REP1 [P6393]; Homo sapiens; RNA-Seq
library_construction_protocol: MSCs total RNA samples of P4, P6 and P12 were separately treated with TRIzol (TAKARA). The extracted total RNA was first removed using a biotin-labeled specific probe (Ribo-Zero™ rRNA Removal Kit) to remove the ribosomal rRNA.After purification, the RNA is fragmented at a certain temperature and ionic environment.A cDNA strand was then synthesized using a random primer and reverse transcriptase in the TruSeq® Stranded kit, and then a double-stranded cDNA was synthesized using DNA polymerase I and RNaseH.During cDNA two-strand synthesis, the RNA template is removed and dTTP is replaced by dUTP.The involvement of dUTP prevents the two strands of cDNA from being amplified in the subsequent process because the polymerase cannot cross the dUTP site on the template as it extends.The double-stranded cDNA product is then linked with an "A" base and connector.The ligation products will be amplified and purified to produce the final cDNA library.Finally, the constructed sequencing library was sequenced. For miRNAs, Experiment Pipeline Steps for Small RNA Sequencing 1) Filter small RNA: separate 18-30nt RNA segment by PAGE gel. 2) 3' adaptor ligation: link 5-adenylated, 3-blocked single-stranded DNA adapter to 3'  end of selected small RNAs from step 1. 3) Reverse primer annealing: the RT primer is added to the solution from step 2, and is cross-linked to the 3' adapter of RNAs and excessive free 3' adapter. 4) 5' adaptor ligation: the 5' adaptor is linked to the 5' end of the product from step 3. The connection (5鈥 adaptor) will be attached to the 5鈥 end only, but it is not c onnected to the 3' adaptor or RT primer hybrid chain. This greatly reduces self-ligation.  5) Strand cDNA synthesis: reverse extend the RT primer in step 3 to synthetize strand cDNA. 6) PCR amplification: use high-ping polymerase to amplify cDNA, enrich cDNA with both 3' and 5' adaptor. 7) Library fragment selection: separate the PCR product of 100~120bp by PAGE gel to eliminate primer-dimer and other products.  8) Library quantitative and pooling cyclization.