GSM4255701: Hmel_rep2; Homo sapiens; OTHER
Culture media was removed, cells were washed with ice-cold PBS containing cycloheximide (0.1mg/ml) and lysed in Lysis Buffer according to the Illumina protocol (RPHMR12126). Ribosomes containing ribosome-protected mRNA fragments (RPFs) were enriched using MicroSpin S-400 columns (GE Healthcare, catalog # 27-5140-01). Ribo-zero rRNA Removal Kit (Illumina, MRZH11124, discontinued) was used to deplete rRNA from RPFs. The entire RPF sample was loaded on a 15% urea-polyacrylamide gel. Gel fragments in 28-32 nucleotide range were cut from the gel. Samples were eluted from the gel overnight at 4°C. Subsequently, end repair, adapter ligation and reverse transcription were carried out according to the manufacturer's protocol. For the cDNA gel purification, the reverse transcription reaction was loaded on a 10% urea-polyacrylamide gel. The samples were eluted from the gel overnight at room temperature. Subsequently, RPFs were circularized and 5 μl of circDNA was used for library amplification. The number of amplification cycles was determined based on the observed sample quality and expected yield, but usually ranged between 8 and 10 cycles. Following amplification, the library was gel-purified using 4% E-Gel EX Agarose Gel (ThermoFisher G401004) and Zymoclean Gel DNA Recovery Kit (Zymo Research D4007), with 4 volumes of ADB buffer to accommodate 4% agarose gel. The resulting libraries were analyzed for quality using Agilent Bioanalyzer 2100 and sequenced for 51 cycles on the Illumina NextSeq platform, using NextSeq 500 high output kit, V2, 75 cycles.
GEO Accession
GSM4255701