Neb next first strand synthesis reaction buffer

The total RNAs of the apical bud (Bud-1, -2, -3) and the second leaf (SL-1, -2, -3) were used to construct the RNA-seq libraries. The sequencing libraries were generated using the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions. In brief, the mRNA was enriched by oligo(dT) magnetic beads and was then cut into short fragments using the NEBNext First Strand SynthesisReaction Buffer (NEB). The first strand cDNA was synthesized using the Random hexamerprimers and M-MuLV Reverse Transcriptase and both DNA polymeraseI and RNase H were used to synthesize the second strand cDNA. After the adenylation of 3′ ends of the DNA fragments, hybridization was performed for the NEBNext Adaptor (NEB), which contained a hairpinloop structure. The AMPureXP system (Beckman Coulter) was used to size-select the cDNAs. The size selected cDNAs were incubated with the USER Enzyme (NEB) and were then amplified using Phusion High-Fidelity DNA polymerase and primers. The quality of the libraries was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). The RNA-seq was performed on the Illumina Hiseq X Ten platform based on the Paired-End 150 (PE150) strategy by Biomarker Tech (Beijing China).

Construction of the cDNA libraries and RNA-Seq were performed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). First, mRNA was purified from 1.5 μg of total RNA from salivary gland tissue using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperatures in NEB Next First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Subsequently, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After the adenylation of 3’ ends of DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments that were preferentially 150~200 bp in length. Then, 3 μL of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system.

Total RNA was obtained from sham group, BLM‐treated group and astilbin‐treated group, respectively. mRNA was purified from total RNA using poly‐T oligo‐attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). These samples were used for sequencing. Sequencing libraries were generated using NEBNext^® Ultra^™ RNA Library Prep Kit for Illumina^® (#E7530L, NEB) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. Following quantile normalization of the raw data, circRNA with at least two out of two present or marginal flags were selected for further data analysis. Differentially expressed circRNAs were identified through fold change filtering.