title: RNA-Seq of 2 ug/mL oligomer treatment sample 3
design: Whole mRNAseq libraries were generated by Guangdong Magigene Biotechnology Co.,Ltd. (Guangzhou, China) using NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) following manufacturers recommendations. Briefly, the bacterial and archaeal 16S and 23S ribosomal RNA (rRNA) transcripts in total RNA samples were reduced by Ribo-zero rRNA Removal Kit. Fragmentation was carried out using NEB Next First Strand Synthesis Reaction Buffer. The first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). When synthesizing the second strand of cDNA, a chain-specific library is constructed by replacing dTTP with dUTP to greatly improving the accuracy of the results. 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 of preferentially 150~200 bp in length, the fragments were selected with AMPure XP beads (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified with AMPure XP beads and library insert size was assessed on the Agilent 2100 system (Agilent Technologies, Waldbron, Germany).
library_name: treatment group 3