GSM4202141: WT rep2; Mus musculus; OTHER
In order to generate a pool of endogenous mouse embryo mRNAs as RNA input for the ribosome-IP, up to 10 stage 11.5 embryos per FVB female were harvested as described in the mouse section, individually collected in 2 ml Eppendorf tubes, washed once with 1x PBS (Gibco, 14190-250), and lyzed in 1 mL TRIzol (Invitrogen, 15596) by pipetting and vortexing, and addition of another 800 µL TRIzol. Embryo lysates were stored at -80°C until total RNA extraction. From each embryo, 150-200 µg total RNA was obtained. From total RNA, poly(A) mRNA was isolated on oligo(dT) beads using the Oligotex mRNA Mini Kit (QIAgen, 70022) or Poly(A) Purist MAG kit (Invitrogen, AM1922) according to the manufacturer's instructions, which yielded ~5 µg mRNA (2-3%) of 150-200 µg total RNA per embryo. Purified embryo mRNA was fragmented to 100-200 nt RNA fragments using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S). Fragmentation was optimized for time and RNA input amount monitoring RNA size using the mRNA Pico Chip (Agilent) on a Bioanalyzer (Agilent), and by 8% denaturing urea-PAGE and SYBR Gold staining. Fragmentation of 1 µg mRNA aliquots for 5 min at 94°C in 1x Fragmentation Buffer (NEB) was optimal to obtain a pool of 100-200 nt fragments. Reactions were quenched on ice and by addition of 1x Stop Solution (NEB). Immediate isopropanol-based precipitation recovered 75-95% of input mRNA as mRNA fragments in water. For Flag-pulldown of Flag-tagged yeast 40S, powderized yeast lysates of a 250 mL culture per three samples were dissolved in 500 µL lysis buffer and the tube was washed with another 200 µL lysis buffer. Lysates were cleared by centrifugation for 5 min at 17,000 g at 4°C and 2 min at 17,000 g at 4°C, and 800 µL lysate was recovered. RPS2-Flag tagged 40S ribosomes were immuno-precipitated by addition of 50 µL washed anti-Flag M2 affinity agarose gel (Sigma Aldrich, A2220-5mL) and 5 µL RNasin Plus (Promega) per sample to 800 µL lysate and 1.5-2 hours of rotation at 4ºC. Beads were washed 3 times with 500 µL lysis buffer and bound ribosomes were resuspended by addition of 200 µL lysis buffer. 10 µg fragmented mRNA from E11.5 FVB embryos in 40 µL per sample were pooled for 6 samples. 5 µL was saved as an input RNA sample for sequencing. Pooled mRNA was refolded in lysis buffer in a total volume of 600 µL as described in the 4xS1m pulldown section and used as input for 6 samples. 10 µg refolded RNA in 100 µL was added to 100 µL ribosome-coupled 50% beads, 3 µL RNasin (Promega) and 100 µL lysis buffer for a total volume of 300 µL for IP by rotation for 2 hours at 4ºC. Bound ribosome-mRNA fragment complexes were rinsed once with 1 mL lysis buffer and washed 3 times with wash buffer for 5 min tumbling at 4°C. Samples were then eluted off the anti-Flag beads using competitive 500 µg/mL 3xFlag peptide (Sigma-Aldrich, F4799-4mg) elution in 150 µL lysis buffer by rotation for 1 hour at 4°C. 5% of the elution was used for protein analysis by WB, and 95% was subjected to TRIzol total RNA extraction and library preparation. Library Preparation and Deep Sequencing 5 µg total RNA isolated from Flag elution samples were treated with Yeast RiboZero Gold (Illumina, MRZY1306) according to the manufacturer's instructions to remove yeast rRNAs from the samples. From the remaining fragmented RNA in water (10 µL, yield 80-160 ng RNA), 30 ng of elution and mRNA fragment input samples were used for library preparation. Library preparation for deep sequencing was performed using the NextFlex Rapid Directional qRNA-Seq Library Prep Kit (Perkin Elmer, Bioo Scientific, NOVA-5130-01D) according to the manufacturer's instructions using 7 unique barcodes. In brief, the standard protocol was applied with the following changes: the initial fragmentation step was omitted and PCR amplification was performed using 16 cycles. DNA fragments were purified for Illumina sequencing, subjected to analysis using the High Sensitivity DNA Assay (Agilent) on a Bioanalyzer (Agilent) and all DNA libraries were pooled to a final concentration of 4 nM. Sequencing was performed at the Stanford Functional Genomics Facility (SFGF) at Stanford University, on the Illumina NextSeq 550 instrument, using 2x 75 nt paired-end sequencing and the following library design: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNT-insert-NNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACBBBBBBBBATCTCGTATGCCGTCTTCTGCTTG, where N is the 2x 8 nt unique molecular index, and B is the 8 nt sample barcode.
GEO Accession
GSM4202141