Nebnext multiplex small rna library prep set

The sequencing library was prepared by following the procedure described in NEBNext Multiplex Small RNA Library Prep Set for Illumina. Briefly, 6 μL of the RNAs (with an effective mRNA input approximately 10¹² molecules per preparation) were ligated to 10x concentrations of the multiplex 3′ SR adaptor (5′-rAppAGATCGGAAGAGCACACGTCT-NH₂-3′) and the 5′ SR adaptor (5′-rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC-3′), followed by reverse transcription. The cDNA constructs were then amplified using PCR by modification of the extension time at 62 °C to 16 s and the number of cycles to 15. The PCR amplified cDNA constructs were purified using a QIAquick PCR Purification Kit followed by gel chromatography using 6% native PAGE (Supplementary Fig. S11). The bands of the proper size for the cDNA constructs were excised and eluted overnight with the elution buffer (provided by the kit). The final cDNA constructs were then precipitated with ethanol and resuspended in distilled H₂O. Finally, 1.0 μL of 1.0 nM of the cDNA constructs (around of 6 × 10⁸ molecules) were used for deep sequencing (2G paired-end) on Illumina HiSeq sequencing platform.

A549 cells were treated with EGCG at the concentration of 40 and 100 μM. Untreated cells were used as a control. Cells were harvested for RNA extraction after 24 h of culture. Total RNA was extracted from the untreated and treated A549 cells using TRizol reagent (Takara Bio Inc., Kusatsu, Japan) following the manufacturer’s protocol. The small RNA library construction and deep sequencing were carried out at AgriGenome Labs Pvt. Ltd., Kochi, Kerala, India. NEBNext® Multiplex Small RNA Library Prep Set for Illumina was used for library construction. The raw counts and the normalized files were submitted in NCBI (National Center for Biotechnology Information) by accession number GSE110514.

QC and quantity measurement of the RNA was performed on an Agilent 2100 Bioanalyzer. All RNA samples had OD260/280 ≥ 1.8 and RNA integrity number (RIN) ≥7. Selection of small RNAs for library preparation was performed using gel electrophoresis. Small RNA libraries were prepared using the NEB Next Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs Cat. NEB-E7580S). The libraries were sequenced with one lane on an Illumina NextSeq 500 instrument with 50-bp single-end reads at the Genomics Center of the Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel. Adaptor-only reads and low-quality reads were filtered out in the Illumina BaseSpace environment. RNA-seq datasets generated are available in the SRA repository (BioProject ID PRJNA494326). The raw dataset is available on https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA494326.

Structure probed RNA was fragmented at 95 °C for 3.5 min in alkaline hydrolysis buffer (Ambion). As fragmentation results in 5′OH, and is hence ligation incompatible, it does not interfere with the downstream library preparation process. Fragmented RNA was then purified using RiboMinus concentration module (Life Technologies), using the modified protocol for RNAs that are <200 bases. The RNA was eluted in 12 µl of nuclease free water and concentrated to 2 µl using a vacuum centrifuge. The RNA was then ligated to 5′ adapter from NEBNext Multiplex Small RNA Library Prep Set for Illumina using T4 RNA ligase1 (T4 RNA ligase buffer, 1 mM ATP, 10% PEG, 10% DMSO) at 16 °C overnight. The 5′ adapter ligated RNAs were then purified through a 6% TBE urea PAGE gel and size selected for 50–200 bases. The RNA was then ligated to 3′ adapter, reverse transcribed, and PCR amplified using the NEBNext Multiplex Small RNA Library Prep Set (New England Biolabs) for Illumina using manufacturer’s instructions. Eighteen cycles of PCR amplification were typically performed for each library.

3-6-day old female ovaries were dissected from each experimental genotype and placed directly in Trizol reagent (Invitrogen), and homogenized. For aub genotypes, Illumina small RNA libraries were prepared by Fasteris according to a proprietary protocol that depletes for 2S-RNA. Because the two biological replicates were prepared at different time points (5/13 and 7/13), they were analyzed separately. Small RNA libraries for spn-E and armi genotypes were prepared as described in [60 (link)]. In brief, total RNAs were extracted according to the manufacturer’s instructions, and size fractionated on a 12% polyacrylamide/urea gel to select for 18–30 nt small RNAs. Small RNAs were treated with 2S Block oligo (5’-TAC AAC CCT CAA CCA TAT GTA GTC CAA GCA/3SpC3/-3’), and were subsequently ligated to 3’ and 5’ adaptors, reverse transcribed and PCR amplified using NEBNext Multiplex Small RNA Library Prep Set for Illumina. Small RNA libraries were further purified from a 2% agarose gel and sequenced on a Illumina NextSeq 500 at the University of Houston Seq-N-Edit Core.