First strand synthesis reaction buffer

A total amount of 1 μg RNA per sample was used as initial material for the RNA-seq. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, RNA fragmentation and short RNA strands were carried out by NEBNext First Strand Synthesis Reaction Buffer under elevated temperature. Subsequently, First cDNA strand was synthesized using random hexamer primers and RNA fragments as template. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs (dUTP), DNA polymerase I and RNase H. RNA fragmentation and short RNA strands were carried out by NEBNext First Strand Synthesis Reaction Buffer under elevated temperature. The library fragments were purified and elution with EB buffer, then terminal repair, add poly (A) and adapter were implemented. In order to select cDNA fragments of preferentially 400 bp in length, the library fragments were purified with agarose gel electrophoresis and the UNG (Uracil-N-Glycosylase) enzyme was used to digest second strand of cDNA. PCR was performed, aimed products were retrieved by agarose gel electrophoresis, and the library was completed. The libraries were clustered and sequenced on an Illumina NovaSeq 6000 platform and 150 bp paired end reads were generated.

Illumina sequencing was performed using three tissues (the intestine, brain, and muscle) obtained from three fish in each group. RNA was extracted as previously described and enriched by oligo(dT) beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized by reverse transcriptase; RNA was then digested by RNaseH, and second strand cDNA was synthesized by DNA polymerase I system. After purification and repair, poly(A) was added to double-stranded cDNA and sequencing adapters were connected. AMPure XP beads were used to screen cDNA (250–300 bp) for PCR augmentation. After secondary purification, the library was constructed and quantified by qRT-PCR (≥ 2 nM), followed by sequencing on Illumina HiSeq 4000. Sequencing was performed by Novogene Co. (Beijing, China). Clean reads (> 100 bp) were obtained by removing reads containing adapters or poly-Ns from the raw data. Clean reads were mapped to the reference genome of C. idella with HISAT2 [58 (link)].

PolyA-RNA was purified and enriched from 25 μg HepG2 total RNA by using VAHTS mRNA Capture Beads (Vazyme, N401-01). Enriched polyA-RNA were fragmented with NEBNext First Strand Synthesis Reaction Buffer (5 ×) in 15 μl volume at 94 °C for 9 min. Then the fragments were purified with 2 volumes of VAHTS RNA Clean beads. To enrich the 5’capped fragments, the fragmented RNA was incubated with 2 U Terminator exonuclease (Lucigen, TER51020), 1x Terminator reaction buffer A, and 20 U RRI in 40 μl volumes for 30 °C, 1 h. The RNA was purified by using RNA Clean & Concentrator Kits (ZYMO research, R1013). The terminator-treated RNA was treated with 5 μl Quick CIP (NEB, M0525S) and 1x CutSmart buffer at 37 °C for 30 min, followed by purification by RNA Clean & Concentrator Kits. Then, the eluted RNA was mixed with Cap-Clip Acid Pyrophosphatase (CELLSCRIPT, C-CC15011H) according to the induction to hydrolyze the pyrophosphate bonds of the 5’cap structures. The RNA was purified by RNA Clean & Concentrator Kits and then ligated with the 3’/5’ adapters. Adapters-ligated RNA was reversed-transcribed by using SSIV and nested RT-PCR strategy for library construction.