title: Transcriptional profiling of bovine muscle-derived satellite cells differentiation using RNA sequencing
design: RNA was extracted from MDSCs during different differentiation stages at 0h, 24h and 72h. Cell pellet by the Trizol method (Invitrogen, USA) according to the instructions. RNA was quantified by an NanoDrop 2000 Spectrophotometer (Thermo, USA), and the quality and integrity was assessed by the spectrophotometer 260/280 ratio. Oligo(dT) magnetic beads adsorption was used to purify mRNA, then Oligo(dT) as primer was used to synthesize cDNA. The 5' ends of tags could be generated by two types of Endonuclease: NlaIII or DpnII. The bead-bound cDNA was subsequently digested with restriction enzyme NlaIII, which recognized and cut off the CATG sites. The fragments aparted from the 3' cDNA fragments connected to Oligo(dT) beads were washed away and the Illumina adaptor 1 was ligated to the sticky 5' end of the digested bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site was the recognition site of MmeI, which was a type of Endonuclease with separated recognition sites and digestion sites. It cutted at 17bp downstream of the CATG site, producing tags with adaptor 1. After removing 3' fragments with magnetic beads precipitation, Illumina adaptor 2 was ligated to the 3' ends of tags, acquiring tags with different adaptors of both ends to form a tag library. After linear PCR amplification, fragments were purified by PAGE Gel electrophoresis. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library. At last, the library could be sequenced using Illumina HiSeq™ 2000 or other sequencer when necessary.