alias: GSM2735648
title: GSM2735648: fPmt1Ko_rep3 [small RNA-seq]; Schizosaccharomyces pombe; ncRNA-Seq
library_construction_protocol: Ribosome footprinting was performed essentially as described (Duncan and Mata, 2014; Ingolia et al., 2011). S. pombe cultures were grown at 30°C to an OD600 of 1.0, 150 µg/ml cycloheximide was added to the medium and cells were incubated for further 15 min at 30°C. Cells were harvested and flash-frozen immediately in liquid nitrogen. 15 OD of each sample was lysed by adding 100 µl lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 200µg/ml cycloheximide, 1% triton, protease inhibitor) and 1 g chilled glass beads (0.5 mm diameter). Samples were homogenized using a FastPrep-24 (MP Biomedicals) at level 6 for 13 seconds and subsequently diluted with 400 µl lysis buffer. Two centrifugation steps (1: 5 min, 4°C, 16000xg; 2: 15 min, 4°C, 16000xg) removed glass beads and cellular debris, respectively. For footprint isolation, 400 µl of the supernatants were digested with 4 U DNase I (ThermoScientific) and 800 U of RNase I (Ambion), for 45 minutes at room temperature with gentle shaking. 800 U of RNasin ribonuclease inhibitor (Promega) was added to quench the reaction. The samples were run on a 17.5 - 50% sucrose gradient to isolate monosomes. Footprint monosome RNAs were end-repaired with T4 polynucleotide kinase (TaKaRa), and size selected (28-31 nucleotides) on a 15% polyacrylamide Tris-borate-EDTA-urea gel. Total RNA was extracted from 100 µl lysate using TRIzol (Invitrogen) according to the manufacturers instructions. Small RNA-seq: Small RNA libraries were constructed according to the protocol using NEB NEXT Small RNA library Prep Set for Illumina (Multiplex Compatible) E7330, and size-selected (120-170 base pairs) before sequencing. RNAseq: After quantification of total RNA and quality assessment on an Advanced Analytical Fragment Analyzer using the DNF-472 High Sensitivity RNA Analysis Kit (all Advanced Analytical Tech. Inc; Ankeny Iowa, U.S.A.), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490). Illumina-compatible RNA-libraries were constructed by using the NEBNext Ultra RNALibrary Prep Kit for Illumina (E7530) as well as the NEBNext Multiplex Oligos for Illumina (E7335) following the distributors instruction manuals. Multiplexed RNA libraries were mixed in equilibrium according to their Qubit (Life Technologies) measurements, fragment distribution profiles on the Advanced Anayltical Fragment Analyzer and quantification of library 5'-and 3'- ends by Real Time PCR. For each library, 30 Mio clusters were sequenced in a 1x 75 base mode on a Illumina NextSeq500 instrument according to the manufacturer’s protocol and using the NextSeq 500/550 High Output Sequencing Kit v2 (75 cycles) (Product Code: FC-404-2005).
identifiers.namespace: GEO
identifiers.id: GSM2735648