Hiseq miseq sequencing

RNA from all samples was extracted using Trizol reagent (Invitrogen) after being ground by a homogenizer. RNA was extracted according to the manufacturer’s instructions. The concentration and OD values of extracted RNA were determined using Nanodrop 2000 (Thermo Scientific CA). RNA agarose gel electrophoresis was also performed to examine the integrity of total RNA, and Agilent 2100 was used for RIN.
At least 5 μg of total RNA, with concentration higher than 200 ng/μL and OD values of 1.8–2.2 were used for the following tests. mRNA was enriched using Oligo (dT) beads which can specifically bind to the poly A tail of mRNA. Enriched mRNA was then placed into fragmentation buffer to be randomly digested into 1.5–2 kb fragments. Then mRNA was reverse transcribed to cDNA, followed by double-stranded DNA synthesis. After the double-stranded DNA was blunt-ended using End Repair Mix, Illumina Hiseq/Miseq sequencing was performed.

Total RNA from samples was isolated according to the manufacturer’s instructions, and its quality was tested using a Nanodrop 2000 machine (Thermo Scientific CA) to determine concentration and OD values. The integrity of total RNA was confirmed visually by agarose gel electrophoresis. Polyadenylated mRNA was purified from the total RNA using Oligo-dT beads, which can specifically bind to the ploy A tail of mRNA. Next, mRNAs were randomly digested into 1.5–2kb kb fragments using the Ambion RNA fragmentation kit. Fragmented mRNAs were used as templates to synthesize single-stranded cDNA using random primers, following the synthesis of double-stranded cDNA. Then, template DNA fragments were end-repaired using End Repair Mix, and adenosine was added to the 3’ ends to ligate the adapter. After the enrichment of the cDNA library by 15 cycles of PCR, PCR products were purified by 2% agarose gel electrophoresis. Target DNA bands were extracted using a Qiagen gel extraction kit. After quantification, bridge PCR was conducted to generate different clusters in cBot. Then, Illumina Hiseq/Miseq sequencing was performed.