sRNA-Seq of BEAS-2B-Ctrl
The RNA samples were extracted with Trizol. Nanodrop, Qubit 2.0, the Agilent 2100 bioanalyzer were used respectively to to detect the purity of RNA samples, concentration and integrity, etc., to ensure that the use of qualified samples for sequencing.RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA).RNA concentration was measured using Qubit RNA Assay Kit in Qubit2.0 Flurometer (Life Technologies, CA, USA).RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Library preparation for sRNA sequencing A total amount of 1.5 g RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNextUltra small RNA Sample Library Prep Kit for Illumina(NEB, USA) following manufacturers recommendations and index codes were added to attribute sequences to each sample. Briefly, First of all, ligated the 3 SR Adaptor. Mixed 3 SR Adaptor for Illumina, RNA and Nuclease-Free Water, mixture system after incubation for 2 minutes at 70 degrees in a preheated thermal cycler. Tube was transfer to ice. Then, add 3 Ligation Reaction Buffer (2X) and 3 Ligation Enzyme Mix ligate the 3 SR Adaptor. Incubated for 1 hour at 25C in a thermal cycler. To prevent adaptor-dimer formation, the SR RT Primer hybridizes to the excess of 3 SR Adaptor (that remains free after the 3 ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated. The second, ligated the 5 SR Adaptor. Then, reverse transcription synthetic first chain. Last, PCR amplification and Size Selection. PAGE gel was used to electrophoresis fragment screening purposes, rubber cutting recycling as the pieces get small RNA libraries. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumia) according to the manufacturers instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated.