ck-3
sample not treated, replicate #3
Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina) according to manufacturers instructions. Three micrograms of total RNA were used as initial material for library preparation. Briefly, mRNA was purified using poly-T oligo-attached magnetic beads. Fragmentation of mRNA was conducted using divalent cations under elevated temperature in the Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and SuperScript II. Second strand cDNA was subsequently synthesized using DNA Polymerase I and RNase H. After adenylation of the 3 ends of DNA fragments, Illumina PE adapter oligonucleotides were ligated and the library fragments were purified using the AMPure XP system (Beckman Coulter). DNA fragments with ligated adaptors on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. The products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing libraries were then sequenced at both ends for 150 bp using a Hiseq Xten platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.
ck-3
SUB4684567
ck-3