Trueseq small rna library prep kit

TrueSeq small RNA library prep kit (Illumina San Diego CA, USA) was used for the preparation of small RNA library and the protocol was followed as per manufacturer instruction. The procedure for small RNA library preparation included adapter ligation, reverse transcription, PCR amplification and pooled gel purification to generate a small RNA library. In the RNA sample, 3′ adapter was specifically modified to target miRNAs. After ligation of adapter to each end, the RNA was reverse transcribed to single-stranded cDNA and sequenced for preparation of small RNA library using an Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA). Further, using a DNA specific chip, library construct (1 μl) was loaded on Agilent Technologies 2100 Bioanalyzer for validation of small RNA library. Before the start of the sequencing, all the individual libraries were clustered together in a single lane on an Illumina HiSeq. 2000 platform using TrueSeq Cluster kit V3- cBot-HS (HiSeq) that generated 0–100 bp paired-end reads.

RNA purity and concentration were assessed at each step using Nanodrop, based on the absorbance ratio 260/280 > 2. RNA integrity was evaluated using Agilent 2100 bioanalyzer system. At least 50 ng of r-RNA depleted RNA was used to generate sequencing libraries using the True-seq small RNA library prep kit (Illumina). All libraries were barcoded and sequenced in parallel on a Next-seq platform for 400 million reads to obtain 75 bp single end reads.

Small RNA Library was prepared using TrueSeq small RNA library prep kit (Illumina San Diego CA, USA) according to the manufacturer's instruction. This whole procedure requires adapter ligation, reverse transcription, PCR amplification, and pooled gel purification to generate a library product. For targeting miRNAs having a 3′ hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes, RNA 3′ adapter was specifically modified to target miRNAs. Further, Adapters were ligated to each end of the RNAs and subsequently reverse transcribed to create single-stranded cDNA and sequenced using miRNA sequencing on an IlluminaHiSeq 2000 platform (Illumina, San Diego, CA, USA). By using a DNA specific chip 1 μl of the resuspended library construct was loaded on Agilent Technologies 2100 Bioanalyzer chip for small RNA Library validation. Prior to the sequencing, all the individual libraries were clustered together using TrueSeq Cluster kit V3-cBot-HS (HiSeq) in a single lane on an IlluminaHiSeq 2000 platform that generated 0–100 bp paired end reads.

The small RNA library (sRNA library) was constructed according to the manufacturer’s instruction of TrueSeq small RNA library prep kit (Illumina San Diego CA, USA), and 2.5 ng RNA per sample was used as the initial amount. The T4 RNA Ligase 1 was ligated to the 3′ end of the RNA, followed by the ligation of T4 RNA Ligase 2 (truncated) to the 5′ adapter. After that, the RNA was reverse transcribed to synthesize cDNA. Finally, small RNA libraries were obtained by screening these recovered fragments using gel separation technology. In the construction of lncRNA library (chain-specific library for removal of ribosomal RNA), the epicenter Ribo-ZeroTM kit was used to remove the ribosomal RNA. Then, rRNA-depleted RNA was fragmented and used as a template to construct the cDNA library. The qualities of the libraries were further tested following these steps: 1) Initial quantification was performed using Qubit 2.0, and the insert size of the library was tested using Agilent 2100. 2) The Q-PCR method was used to accurately quantify the effective concentration of the library (effective library concentration > 2 nM). After the libraries’ quality tests were passed, different libraries performed pooling according to the amount of target data, followed by sequencing on the Illumina HiSeq platform.

Following extraction and purification, about 1 μg total RNA per sample was used to construct the small RNA (sRNA) library using TrueSeq small RNA library prep kit (Illumina San Diego CA, USA) according to the manufacturer’s instruction. Adapters were ligated to the 3′ end of the RNA, followed by the ligation of the 5′ adapter. Subsequently, the RNA was reverse transcribed to create single-stranded cDNA, followed by single-end sequencing (50 base pairs in length) on an Illumina on the HiSeq4000 sequencer (Illumina, San Diego, CA, USA).
Raw data (raw reads) were processed with an in-house pipeline consisting of adapter trimming, read alignment and read counting. The trimmed reads, also known as clean reads, were mapped to the human reference genome GRCh38 using the popular alignment tool Bowtie^{4 (link)}. Then, the modified software miRDeep2 (https://www.mdc-berlin.de/8551903/en/) was used to compute miRNA read counts^{5 (link)}. Mature miRNA and miRNA precursors were downloaded from miRBase. Moreover, the differentially expressed miRNAs (DEmiRNAs) between samples were identified using DEGseq package in R. The p-value <0.01 and |log2 (Fold_change)| > 2 were used as the cut-off criteria.