title: GSM3725004: -AA_polysome_rep.1; Homo sapiens; OTHER
library_construction_protocol: Cells were washed and detached with 10 ml ice cold PBS and collected at 1000 rpm for 5 minutes at 4°C. Cells were lysed in 250 µl lysis buffer (100mM KCl, 20mM Tris pH 7.5, 5mM MgCl2, 1mM DTT, 1mM Leupeptin, 1mM Pepstatin, 1mM Aprotinin, 0,1 mM PMSF, 0,1 mM AEBSF, 0,5% NP40, 100 µg/ml cycloheximide, 40 U/ml RNAsin (Promega)) for 10 minutes on ice. Cell debris were pelleted at 10k rpm for 10 minutes in an Eppendorf tabletop centrifuge. Ribosome protected fragments (between 17-34 nts) were purified on a 15% PAA 8M Urea page. First, the ends were repaired using T4 polynucleotide kinase (NEB) with and without ATP to create a phosphorylated 5'- and a dephosphorylated 3'-end for 1 h at 37 °C each. Afterwards, libraries were prepared using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB) following the manufacturer's protocol with only minor modifications. All adapters and primers were diluted 1:3 and 14 cycles of PCR were performed. No further size selection was performed. Libraries were quantified using a Qubit 3.0 (Thermo Scientific) and a 2100 Bioanalyzer (Agilent) and pooled equimolar. The pool of 12 ribosome profiling libraries was sequenced on a High-Output NextSeq 500 (Illumina) sequencing run with a read length of 75 nt.
attributes.tag: GEO Accession
attributes.value: GSM3725004