Nebnext multiplex small rna library prep kit

The total RNA extraction method was the same as that employed for RNA-seq. A total of 1.5 µg of total RNA was used for the construction of libraries using the NEB Next Multiplex Small RNA Library Prep Kit (Illumina Inc., San, Diego, CA, USA) according to the manufacturer’s protocol. The purified RNAs were ligated with 3’ and 5’ adapters for Illumina processing. Reverse transcription followed by PCR was used to create cDNA constructs based on the small RNAs ligated with 3’ and 5’ adapters. DNA fragments of 140–160 bp in length were purified. The cDNA library was recovered for Illumina sequencing library preparation. The small RNA library inserts were 18–30 bp, and the library was sequenced on an Illumina HiSeq 2000 platform.

After total RNA was extracted by the Trizol reagent kit (Invitrogen, Waltham, MA, USA), an NEB Next Multiplex Small RNA Library Prep Kit (Illumina Inc., San Diego, CA, USA) was used for small RNA library construction according to the manufacturer’s instructions. The RNA molecules in a size range of 18–30 nt were enriched by polyacrylamide gel electrophoresis (PAGE). Then, 3′ adapters were added, and the 36–44 nt RNAs were enriched. Next, 5′ adapters were ligated to the RNAs as well. The ligation products were reverse transcribed by PCR amplification, and the 140–160 bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

For the NGS-based sequencing, DRG tissues from 10 randomly selected pairs of CFA-treated and untreated rats were extracted. The RNA-seq library was prepared with NEBNext Ultra RNA with Poly-A selection and was sequenced on an Illumina Hi-Seq 4000. The small RNA-seq library was constructed with NEBNext Multiplex Small RNA Library Prep Kit for Illumina (#7560S). The targeted bisulfite sequencing library was prepared with a DNA library construction TruSeq DNA LT Sample Prep Kit v2 for Illumina. The C-T transition was performed using a C-T transition EpiTect Bisulfite kit from Qiagen (Germany).

MicroRNA was extracted from all 24 samples with an EASYspin plant microRNA extraction kit (Aidlab biotechnologies Co., Ltd, Beijing, China), according to the manufacturer’s instructions. The purity, concentration, and integrity of the microRNA samples were tested using an Agilent Bioanalyzer 2100 (Agilent, Waldbronn, Germany). MicroRNA libraries were constructed using the NEB Next Multiplex Small RNA Library Prep Kit for Illumina. Then, these microRNA libraries were sequenced on an Illumina novaseq6000 at Biomarker company (Beijing, China), and 50 single-end reads were generated. All the raw data were uploaded to SRA (https://dataview.ncbi.nlm.nih.gov/object/PRJNA579002?reviewer=c9ctcl8rfej71udnds5o2jcn0g).

Three micrograms of total RNA per sample was used to construct sequencing libraries with a NEBNext multiplex small RNA library prep kit for Illumina (NEB, USA) following the manufacturer’s protocol. Briefly, the NEB 3′ SR adapter was ligated to the 3′ end of miRNAs, siRNAs (small interfering RNAs), and piRNAs (piwi-interacting RNAs). The SR real-time primer was then annealed to the 3′ SR adapter to initiate double-stranded DNA (dsDNA). The 5′ end adapter was connected to the 5′ ends of miRNAs. First-strand cDNA synthesis with M-MuLV Reverse Transcriptase (RNase H-). The libraries were amplified by 35 PCR cycles with index (X) primer, SR primer for Illumina, and LongAmp Taq 2X master mix. The PCR products were separated on 8% polyacrylamide gel for 80 min at 100 V. DNA fragments responding to 140-160 bp (the length of sRNAs with 5′ and 3′ adapters) were purified from the gel and eluted in 8 µL of elution buffer. Library quality and titer were evaluated using a DNA high sensitivity chip and Agilent Bioanalyzer 2100 instrument. A TruSeq SR Cluster Kit v3-cBot-HS (Illumina) was used for cluster formation on a cBot cluster generation system following the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq 2,500 platform as 50-bp single-end reads.

Quality control of RNA samples was performed using the RNA 6000 Nano Kit (for total RNA) and smallRNA Kit (for small RNA species) on a 2100 Bioanalyzer (Agilent). Sequencing libraries were prepared at the Core facility Genomics, Medical University of Vienna using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina according to the manufacturer’s protocols (New England Biolabs). Libraries were QC checked for appropriate insert size on a Bioanalyzer 2100 using a High Sensitivity DNA Kit and quantitated using Qubit dsDNA HS Assay (Invitrogen). Pooled libraries had an average length of 150 bp and were sequenced on a NextSeq500 instrument (Illumina) in 1 × 75 bp sequencing mode. Reads with a read length > 5 bp were adaptor trimmed and trimmed from the 3′-end to a length of 50 bp using FASTQ Toolkit App from BaseSpace (illumina). The Small-RNA App from BaseSpace was used for read alignment against four reference databases (abundant, mature miRNA, other RNA, and genomic). The outputs contain hits to mature miRNAs, isomiRs, and piRNAs expressed as ‘counts’.