GSM4711302: depol 0 hours 2 [k0h2.small.rna]; Homo sapiens; miRNA-Seq
Cells were exposed to cycloheximide (0.1mg/mL) prior to detergent lysis and scraping as per the Illumina TruSeq Ribo Profile kit manual. Production of ribosome protected mRNA fragments (RPFs) and library preparation were then performed using the Illumina TruSeq Ribo Profile kit (H/M/R), with minor amendments to the manufacturer's protocol. Briefly, 300µL clarified lysate was treated with 9µL RNase I (45 min) to digest unprotected RNAs, after which reactions were stopped with 15µL SUPERase inhibitor (Invitrogen). Small RNA fragments were then enriched using MicroSpin S-400 columns (GE Life Sciences), prior to ribosomal RNA depletion, RPF size selection, end repair, adapter ligation, reverse transcription and PAGE purification, conducted according to the manufacturer's instructions. RPF cDNA amplicons were subsequently produced with 12x PCR cycles, adapter dimers were removed via 8% native polyacrylamide gel and samples were quantified via Agilent high sensitivity DNA chip. For sequencing, samples were pooled in equal volumes and concentrations, denatured with NaOH (0.2mM) and diluted to a final concentration of 1.8pM in Tris-HCl (200mM, pH 7) and HT1 buffer. RPF libraries were then subjected to 76 single-end sequencing cycles using the Illumina NextSeq 500 benchtop sequencer.
GEO Accession
GSM4711302