title: GSM2549652: siPKM_RPF; Mus musculus; OTHER
library_construction_protocol: For ribosome profiling experiments, extraction was performed as described before with modifications below (PMID: 22836135). Main modifications are the use of RNase A/T1 instead of RNase I for ribosome footprinting and the use of Ribo-Zero Gold rRNA removal kit. CHX-treated cells were lysed using polysome lysis buffer without SUPERase In RNAse Inhibitor (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM DTT, 8% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 100 μg ml−1 CHX, 25 U ml-1 TurboDNase (25 U ml-1 Ambion, cat. no. AM2238), Complete Protease Inhibitor EDTA-free (Sigma-Aldrich, cat. no. 11836170001), ProteCEASE EDTA-free (G biosciences, cat. no. 786-328) in nuclease-free water (Invitrogen)). RNA for RNA-Seq was isolated using TRIzol (Invitrogen) following manufacturer’s instructions. For ribosome profiling, after lysis, nuclei were removed by three consecutive centrifugations (1000g, 5 min at 4 °C). RNA amount is measured using UV spectrophotometry (Nanodrop ND-1000 spectrophotometer, ThermoScientific) and per ~ 600 μg RNA, 1 μg RNase A (Invitrogen, cat. no. AM2270) and 2000 U RNase T1 (Invitrogen, cat. no. AM2280) were used to footprint ribosomes for 30 minutes at RT. RNase A/T1 digestion was quenched with 180 U SUPERase In RNAse. Ribosomes were enriched by adding the RNAse digested lysate onto 1 M sucrose cushion in lysis buffer and centrifuged in a TLA100.3 rotor (Beckman) for 4 h at 70,000 rpm at 4 °C. The ribosome pellet containing the ribosome footprinted Ribo-Seq library was washed briefly in nuclease-free water and resuspended further in TRIzol (Invitrogen). For FAST-iCLIP, experiments were performed as described previously with the specific modifications below (PMID: 25411354). Whole-cell lysates were generated in iCLIP lysis buffer (50 mM HEPES, 200 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40, 0.2% Triton X-100, 0.5% N-lauroylsarcosine) and briefly sonicated using a probe- tip Branson sonicator to solubilize chromatin. Each iCLIP experiment was normalized for total protein amount, typically 1 mg, and partially digested with RNase I (ThermoFisher Scientific, cat. no. AM2294) for 10 min at 37°C and quenched on ice. PKM2-FLAG-HA was isolated with anti-FLAG agarose beads (Sigma-Aldrich) for 1 h at 4°C on rotation. Samples were washed sequentially in 1 ml for 5 min each at 4°C: 2× high stringency buffer (15 mM Tris-HCl, pH 7.5, 5 mM EDTA, 2.5 mM EGTA, 1% Triton X- 100, 1% sodium deoxycholate, 120 mM NaCl, 25 mM KCl), 1× high salt buffer (15 mM Tris-HCl pH 7.5, 5 mM EDTA, 2.5 mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 1 M NaCl), 1× NT2 36 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40). Purified PKM2-FLAG- HA was then eluted off anti-FLAG agarose beads using competitive FLAG peptide elution. Each sample was resuspended in 500 μl of FLAG elution buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.5% NP- 40, 0.1% sodium deoxycholate, 0.5 mg ml−1 Flag peptide) and rotated at 4°C for 30 min. The Flag elution was repeated once for a total of 1 ml elution. PKM2-FLAG-HA was then captured using anti-HA agarose beads (Pierce) for 1 h at 4°C on rotation. Samples were then washed as previously in the anti- FLAG agarose beads. For ribosome profiling experiments, library preparation steps were adapted from a previous protocol (PMID: 22836135) and the ARTseq Ribosome Profiling Kit manual (Epicentre, cat. no. RPHMR12126). After extraction and precipitation, both ribosome footprinting and RNA-Seq libraries were depleted of rRNA using the Ribo- Zero Gold rRNA Removal Kit (H/M/R) (Illumina, cat. no. MRZG126) following manufacturer’s instructions except that the 50 °C incubation step was omitted. Samples were then purified using Zymo Research RNA Clean and Concentrator-5 columns (Zymo Research, cat. no. R1016) as described in the ARTseq protocol. Libraries were then end repaired and ligated to linker as described previously (PMID: 22836135) with modified Zymo Research RNA Clean & Concentrator-5 column purification after each step (100 μl sample, 200 μl RNA Binding Buffer, 450 μl ethanol) instead of isopropanol precipitation. The RNA-Seq and Ribo-Seq libraries were sequenced on the Illumina NextSeq 500 with 75 bp single reads using the high output kit. For FAST-iCLIP experiments, preparation of UV-crosslinked material was prepared as per the FAST- iCLIP protocol (PMID: 25411354). Final library material was quantified on the BioAnalyzer High Sensitivity DNA chip (Agilent) and then sent for deep sequencing on the Illumina NextSeq machine for single-end 75-bp cycle run. RNA-Seq, Ribo-Seq, iCLIP-Seq
attributes.tag: GEO Accession
attributes.value: GSM2549652