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Sample GSM6470183

Query DataSets for GSM6470183

Status

Public on Feb 11, 2023

Title

R2_RNS_siABC

Sample type

SRA

Source name

HeLa

Organism

Homo sapiens

Characteristics

cell line: HeLa
sirna: PRRC2A + PRRC2B + PRRC2C
fraction: Total RNA
antibody: -

Treatment protocol

Cells were transfected with Lipofectamine RNAimax for siRNA mediated knockdowns. We seeded cells at 200.000 cells per 6-well, transfected them with 3 µl 10 µM siRNA (or siRNA Tetraplex) and 9 µl Lipofectamine reagent. We then re-seeded cells for experiments 72 hours post transfection. Sequences of siRNAs are provided in the publication.

Growth protocol

We cultured HeLa cells in DMEM +10% fetal bovine serum (FBS) +100 U/ml Penicillin/Streptomycin (Gibco 15140122). We sub-cultured the cells using Trypsin-EDTA. HeLa cells were tested negative for mycoplasma and authenticated using SNP typing.

Extracted molecule

total RNA

Extraction protocol

Selective 40S and 80S ribosome footprinting was carried out as published in (Bohlen et al., 2020a; Bohlen et al., 2020b) [Nature Protocols] and as briefly below.
Preparation of non-denaturing cell lysates: We seeded Hela cells at 1.5 million cells per 15 cm dish in 20 ml growth medium two days before cell harvest. For cell harvest, we poured off growth medium and washed cells quickly with ice-cold washing solution (1x PBS 10 mM MgCl2, 800 µM Cycloheximide). After quickly pouring off the washing solution, we added freshly prepared crosslinking solution (1x PBS, 10 mM MgCl2, 0.025% PFA, 0.5 mM DSP, 200 µM Cycloheximide) to the cells. We incubated cells with crosslinking solution for 15 minutes at room temperature while slowly rocking. We poured the crosslinking solution off and inactivated the remaining crosslinker for 5 minutes with ice-cold quenching solution (1x PBS, 10 mM MgCl2, 200 µM Cycloheximide, 300 mM Glycine). After removal of the quenching solution we added 150 µl of lysis buffer (0,25 M HEPES pH 7.5, 50 mM MgCl2, 1 M KCl, 5% NP40, 1000 μM Cycloheximide) to each 15 cm dish, resulting in 750µL of lysate. Lysis was carried out at 4°C. Then we scraped cells off the dish and collected the lysate. After brief vortexing, we clarified lysates by centrifugation at 20.000xg for 10 minutes at 4°C.
Sucrose gradient centrifugation: We collected Supernatant and determined the approximate RNA concentration using a Nanodrop photo-spectrometer. We added 100 U of Ambion RNase 1 per 120 µg of measured RNA. To obtain polysome profiles, we did not add RNase. We incubated the Lysates for 5 minutes at 4°C and then loaded them onto 17.5%-50% sucrose gradients and centrifuged them for 5 hours at 35.000 rpm in a Beckman Ultracentrifuge in the SW40 rotor. Then we fractionated Gradients using a Biocomp Gradient Profiler system. We collected 40S and 80S fractions for immunoprecipitation and footprint isolation. We used 40S and 80S fractions corresponding to roughly one or two 15 cm dishes for direct extraction of RNA for total footprint sample and we used 40S and 80S fractions corresponding to roughly ten 15 cm dishes for immunoprecipitation of initiation factor bound ribosomes, to these fractions, we added NP40 to 1% final concentration.
Immunoprecipitation: For immunoprecipitation, we bound antibodies to protein A or protein G magnetic dynabeads (Thermo) according to the manufacturer’s instructions. For PRRC2C selective ribosome footprinting, we used 40 µl antibody (Biomol #A303-315A) for each the 40S and 80S immunoprecipitation. We washed Beads three times and then added them to the cell lysates or 40S or 80S fractions. We incubated Solutions with beads for 2 hours or over-night, rotating at 4°C. Then we washed the beads three times with bead wash buffer (20 mM Tris pH 7.4, 10 mM MgCl2, 140 mM KCl, 1% NP40), including a change of reaction-vessel during the last wash. We increased the Bead volume to ~500µl with bead wash buffer. Then we subjected total footprint fractions and IPed fractions to crosslink removal and RNA extraction: we added 55 µl (1/9th of volume) of crosslink-removal solution (10% SDS, 100 mM EDTA, 50 mM DTT), 600 µl Acid-Phenol Chloroform (Ambion) and incubated the mixture at 65°C, 1300 rpm shaking for 45 minutes. We then placed Tubes on ice for 5 minutes, spun for 5 min at 20.000 g and washed the supernatant once with acid-phenol chloroform and twice with chloroform. Then we precipitated RNA with Isopropanol and subjected it to library preparation (see below).We used the organic phase to isolate the precipitated or total proteins. We added 300 µl of ethanol, then 1,5 ml of isopropanol and incubated the solutions at -20°C for 1 hour. We then sedimented the precipitated proteins by centrifugation at 20.000g for 20 minutes, washed them twice with 95% ethanol 0,3 M Guanidine HCl, dried and resuspended them in 1x Laemmli buffer.
Deep-sequencing library preparation: After RNA extraction from total and IP-purified fractions, we determined the RNA quality and integrity on an Agilent Bioanalyzer using the total RNA Nano 6000 Chip. For size selection, we ran the RNA on 15% Urea-Polyacrylamide gels (Invitrogen) and excised fragments of size 25-35 nt (80S libraries) and 20-80 nt (40S libraries) using the Agilent small RNA ladder as a reference. We extracted RNA from the gel pieces by smashing the gels into small pieces with gel smasher tubes and extracting the RNA in 0.5 ml of 10 mM Tris pH 7 at 70°C for 10 minutes. We removed Gel pieces and precipitated RNA using isopropanol. We then dephosphorylated the Footprints using T4 PNK (NEB) for 2 hours at 37°C in PNK buffer without ATP. Then we again precipitated and purified the Footprints using isopropanol. For 40S footprints, we depleted contaminating 18S rRNA fragments as follows. We used prevalent 18S rRNA fragments from the first round of 40S footprinting to design complementary Biotin-TEG-DNA oligonucleotides (sequences listed in (Bohlen et al., 2020a)), ordered from Sigma-Aldrich). We then hybridized 100ng of RNA footprints to a mixture (proportional to occurrence of the fragment) of these DNA oligos (in 40x molar excess) in (0.5M NaCl, 20mM Tris pH7.0, 1mM EDTA, 0.05% Tween20) by denaturing for 90 sec at 95°C and then annealing by reducing the temperature by -0.1°C/sec down to 37°C, then incubating 15min at 37°C. We pulled out Hybridized species using Streptavidin magnetic beads (NEB) by incubating at room temperature for 15 minutes, and purifying the remaining RNA by isopropanol precipitation. We then assayed Footprints using an Agilent Bioanalyzer small RNA chip and Qubit smRNA kit. We used 25 ng or less of footprint RNA as input for library preparation with SMARTer smRNA-SeqKit for Illumina from Takara / Clontech Laboratories according to the manufacturer’s instructions. We sequenced Deep-sequencing libraries on the Illumina Next-Seq 550 system.
For RNA-seq libraries, we extracted total cell RNA using TRIzol and performed library preparation using the Illumina TruSeq Stranded library preparation kit. We also sequenced these RNA-seq libraries on the Illumina Next-Seq 550 system.
Polyadenylation-based, oligo dT primer Reverse transcription, followed by PCR amplification.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

Total RNA

Data processing

Adapter sequences and randomized nucleotides (Nextflex) or polyA stretches (Clonentech/Takara) were trimmed from raw reads using cutadapt (https://doi.org/10.14806/ej.17.1.200). Nuclear and mitochondrial Ribosomal RNA and tRNA reads were removed by alignment to human tRNA and rRNA sequences using bowtie2 (22388286). Then, the remaining reads were separately aligned to the human transcriptome (Ensembl transcript assembly 94) and human genome (hg38) using BBmap (sourceforge.net/projects/bbmap/). Multiple mappings were allowed. Secondary mappings were counted when analyzing single transcripts, but not counted in metagene plots to avoid biasing genes with many transcript isoforms or reads with low sequence complexity. Metagene plots, single transcript traces and grouped analyses were carried out or created with custom software written in C, supplied in Supplemental Dataset 1. Read counts for metagene plots of whole transcripts that encompass 5’UTRs, ORFs and 3’UTRs were normalized for the length of each of these features to make them comparable. 70 of the 41.314 transcripts were excluded from the analyses because PCR artefacts mapped to these transcripts. 2-D metagene plots were visualized using Fiji (Schindelin et al., 2012). The same, linear color gradient was always used to indicate read counts at each position in these 2-D metagene plots. Importantly, the maximum intensity of the color gradient was adjusted for the library size of the sample displayed so that 2-D metagene plots can be compared across panels. For 80S plots, only reads with footprint lengths between 26 to 37 nt were counted. For 5’UTR plots, only 40S footprints larger than 29 nt were counted because of the occurrence of artefacts and misaligned reads in the shorter population.
Translated uORFs were defined by the presence of any 80S ribosome footprints in a 10-nucleotide window around the uORF start codon. Only ATG initiated uORFs were considered. uORFs annotated by uORF-Tools (31513641) were downloaded from supplemental table 2 and filtered for uORFs with an intercistronic space > 80 nt.
Assembly: Ensemble transcript assembly 94
Supplementary files format and content: Excel files: Tables of the raw counts for each ENSEMBL transcript in each sample counted in coding sequence (80S and RNA-seq) or 5'UTR (40S).

Submission date

Aug 17, 2022

Last update date

Feb 11, 2023

Contact name

Jonathan Bohlen

Organization name

DKFZ

Department

B140

Lab

AG Teleman