Ampure xp

RNA-seq libraries of 4-TU labeled samples were prepared using the ScriptSeq V2 RNA-Seq Library Preparation Kit (cat. SSV21124, Illumina, San Diego, CA) following the manufacturer’s protocol. In brief, cDNA was synthesized using ScriptSeq cDNA synthesis primer, StarScript AMV reverse transcriptase, and 100 mM DTT. The 3′ end was tagged using Terminal tagging premix and DNA polymerase. cDNA was purified using 1.8× AMPure XP, and libraries were PCR amplified by adding Illumina adapter sequence and an index in the library. Final libraries were again purified with 1× Agencourt AMPure XP (cat. A63881, Beckman Coulter, Brea, CA) and libraries were validated using Qubit 2.0 (Thermo Fisher, Waltham, MA) and High sensitivity DNA bioanalyzer (Agilent, Santa Clara, CA). Libraries were diluted to 2 nM for sequencing into HiSeq-2500 (Illumina, San Diego, CA).

Genomic DNA (1–5 μg) was sheared to 200–500 bp by Covaris S220 sonicator (Covaris, USA). End repair was then performed following the manufacturer’s instructions (End-It DNA end repair kit, Epicentre, USA). After Ampure XP (Sigma, USA) purification, adenine was added to the 3′ end with 3 μl DNA Taq polymerase (M0267S, NEB) and 1 mM dATP in 50 μl reaction solution incubated at 70 °C for 30 minutes. After Ampure XP purification, 1 μl of Illumina Trueseq adaptors were ligated with 4 μl T4 DNA ligase (M0202L, NEB) in 40 μl reaction solution and incubated at 16 °C overnight. Adaptor-ligated DNA fragments of 300–600 bp were collected from 2 % agarose gel, and then bisulfite-treated using an Imprint DNA modification Kit (MOD50-1 KIT, Sigma, USA) according to the manufacturer's instructions. To ensure conversion of uracil to thymine and avoid the potential PCR-induced bias [55 (link)], we minimized PCR cycles (6–10 cycles) to amplify the libraries, and fragments of 300–600 bp were then collected using 2 % agarose gel electrophoresis.

Total RNA was extracted using Biozol reagent (Bioer, Hangzhou, China), and was then visualized on 1% agarose gel. The RNA quality was estimated using a NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA) and the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA samples with an RNA integrity number (RIN) higher than 8.0 were qualified. The RNA quantity was determined using the Qubit^® RNA assay kit (Life Technologies, CA, USA).
In order to construct sequencing libraries, mRNA was enriched by treating total RNA with the Epicentre Ribo-zeroTM rRNA removal kit (Epicentre, USA). Sequencing libraries were constructed using NEBNext^® Ultra^TM directional RNA library prep kit for Illumina^® (NEB, USA), according to the manufacturer’s instructions. Next, DNA fragments were purified with AMPure XP (Beckman Coulter, Beverly, USA) and treated with 3 μl of USER enzyme (NEB, USA) at 37 °C for 15 min. DNA fragments were then amplified using Phusion High-Fidelity DNA polymerase, universal PCR primers, and index (X) primers. Finally, PCR products were purified using AMPure XP and their quality was evaluated using the Agilent Bioanalyzer 2100.
Index-coded samples were clustered on a cBot cluster generation system with a HiSeq. 4000 PE cluster kit (Illumina). Afterwards, DNA libraries were sequenced on the Illumina Hiseq 4000 platform.