title: GSM6514325: belt_deletion; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: Illumina adapter sequences were incorporated to the libraries using PCR. We collected 145 C. elegans pri-miRNAs from MirGeneDB v2.0. We omitted 7 duplicated pri-miRNAs sharing the same miRBase ID. Cel-pri-mir-229 were excluded since its length is longer than 150 nt.   The C. elegans pri-miRNAs were pooled into a substrate mixture.  To clone the cleaved products, the gel pieces containing cleaved products sizing from 50 - 70 nt were sliced out, gel purified, precipitated by IPA, 80% ethanol washed and air dried. The RNAs were dissolved in 10 μL distilled water and ligated with 10 pmol of 3ꞌ-adapter (4N-RA3, /5rApp/NN NNT GGA ATT CTC GGG TGC CAA GG/3ddC/). The ligation was performed in a total 20 μL reaction containing 1 μL  T4 RNA Ligase 2, truncated KQ (NEB, M0373L), 2 μL 10x T4 RNA ligase buffer, 6 μL of 50% PEG8000, and 0.5 μL of SUPERase•In RNase inhibitor (20 U/μL). The mixture was incubated at 25°C for 16 h. After that, it was treated with 20 μL 2x TBE-Urea sample buffer, denatured at 75 °C for 5 min, and loaded onto a pre-run 12% Urea-PAGE. The 3ꞌ-adapter ligated RNAs were sliced and gel purified. The ligated RNAs were dissolved in distilled water and subjected to the 5ꞌ-adapter ligation reaction. For this second ligation reaction, 10 pmol of 5ꞌ-adapter (RA5-4N, GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN), 1 μL T4 RNA ligase I (NEB, M0204L), 2 μL 10x T4 RNA ligase buffer, 6 μL of 50% PEG8000, and 0.5 μL of SUPERase•In RNase inhibitor (20 U/μL) were used. The reaction was kept at 25°C for 16 h and directly reverse transcribed with R-RA3 primer (TTG GCA CCC GAG AAT TCC A) using Superscript IV reverse transcriptase (Invitrogen, 18090050). The resulting cDNA was PCR amplified using a pair of sequencing primers RP1 (AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA), and each of the specific primers, RPIx.   To clone the uncleaved substrate, 10 pmol of the substrate mixture was ligated with 30 pmol of  3ꞌ-adapter as described above. The ligated RNA was reverse transcribed with the cirRTP primer (/5Phos/NNN NNN GA TCG TCG GAC TGT AGA ACT CTG AAC /iSp18/CC TTG GCA CCC GAGAAT TCC A) using Superscript IV reverse transcriptase (Invitrogen, 18090050). The cDNA was treated with 0.1mM NaOH at 95°C for 5 mingel to eliminate the RNAs and gel eluted. The purified cDNA was circularized using the CircLigase™ ss- DNA Ligase (Epicentre, CL4115K) and loaded onto a pre-run 18% Urea-PAGE. The circular DNA were gel purified and PCR amplified using sequencing primers RP1 (AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA), and a specific primer, RPIx. The resulting DNA libraries were sequenced with  Illumina NovaSeq 6000 system in 150 bp paired-end mode.