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Sample GSM4026934

Query DataSets for GSM4026934

Status

Public on May 09, 2021

Title

Rep2_RNA_Cre_S6

Sample type

SRA

Source name

E10.5 mouse forelimbs

Organism

Mus musculus

Characteristics

genotype: Prx1Cre;Rps6lox/+;Trp53+/+
genotype simplified: Rps6 het; Trp53 WT
replicate: Replicate 2

Growth protocol

Embryos were harvested at E10.5 from Prx1Cre x Rps6lox/lox;Trp53+/- mice

Extracted molecule

total RNA

Extraction protocol

Limb pairs from each embryo were lysed via vigorous pipetting and 30 minute incubation at 4oC in 215 μL buffer A (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM DTT, 8% glycerol, 1% Triton X-100, 100 μg/ml CHX (Sigma-Aldrich, C7698-1G), 20 U/ml Turbo DNAse (Thermo Fisher, AM2238), and Complete Protease Inhibitor EDTA-free (Sigma-Aldrich, 11836170001). Lysates were cleared via sequential centrifugation at 1,300g, 5 min and 10,000g, 10 min at 4oC. For RNA input for RNA-Seq, 70 μL of lysate was diluted in 55 μL of water and stored at -80oC in 375 μL of TRIzol LS (Thermo Fisher, 10296010). For ribosome profiling, 120 μL of cleared lysate was treated with 0.5 μg RNase A (Thermo Fisher, AM2271) and 300 U RNase T1 (Thermo Fisher, EN0541) for 30 min, RT with gentle rocking. The reaction was stopped by the addition of 100 U SUPERase RNase Inhibitor (Thermo Fisher, AM2694). Ribosomes were enriched by adding 110 μL lysate onto 900 μL sucrose cushion buffer (1 M sucrose in buffer A containing 20 U/mL SUPERase RNase Inhibitor), and centrifuging in a TLA 120.2 rotor (Beckman) 70,000 rpm, 4 h, 4 °C. The ribosome pellet containing the ribosome footprinted Ribo-Seq library was resuspended in 500 μL TRIzol.
Library preparation was adapted from previous protocols59,61 and the ARTseq Ribosome Profiling Kit manual (Epicentre, Illumina). In summary, total RNA and ribosome footprints were extracted using the Direct-zol Micro Kit (Zymo, R2060) with in-column DNase I treatment. To have an adequate amount of RNA for subsequent steps, pairs of samples of the same genotype were pooled to generate a single replicate sample, brought up to 90 μL with water, and precipitated with 150 μL isopropanol overnight -80oC after addition of 10 μL 3 M NaOAc pH 5.5 and 1.5 μL 15 mg/mL GlycoBlue Coprecipitant (Thermo Fisher, AM9515). The samples were then centrifuged at 21,000g, 30 min, 4oC, supernatant was removed, and the RNA pellet was washed 500 μL cold 75% ethanol. Pellets were dried for 10 min, RT and resuspended in 15 μL nuclease free water. After extraction and precipitation, both ribosome footprinting and total RNA samples were depleted of rRNA using the Ribo-Zero Gold rRNA Removal Kit (H/M/R) (Illumina, catalog no. MRZG126) with the modification that the 50oC incubation was not performed for the ribosome footprinting samples. The samples were then column purified (RNA Clean & Concentrator 5, Zymo Research, catalog no. R1016) with the modifications that 2 volumes of RNA Binding Buffer and 4.5 volumes of ethanol were added to ribosome footprinting samples to purify small RNAs and 1 volume of RNA Binding Buffer and 1 volume of ethanol was added to total RNA samples to isolate RNA > 200 nt. Total RNA samples were then fragmented by partial alkaline hydrolysis. The samples were diluted to 100 μL with 5 mM Tris-HCl, pH 7.5 and incubated with 100 μL 2x alkaline fragmentation buffer (100 mM Na2CO3 pH 9.2, 2 mM EDTA) for 20 minutes at 95oC. The reaction was neutralized with 440 μL STOP Buffer (70 μL 3 M NaOAc pH 5.5, 2 μL Glycoblue, and 370 μL nuclease free water) and isopropanol precipitated overnight at -80oC. Ribosome protected fragments and total RNA samples were then size selected by running the samples out on a 15% TBE-Urea polyacrylamide gel. Ribosome protected fragments were size selected between 28-nt and 34-nt as marked by RNA oligonucleotides oNTI199 and oNTI265, respectively62. Total RNA samples were size selected between 34-70 nt as marked by a 10 bp DNA ladder (Invitrogen, catalog no. 10821015). Gel slices were crushed and extracted at room temperature overnight in 400 μL RNA extraction buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.25% SDS) and isopropanol precipitated. Samples were then 3’dephosphorylated by denaturing at 65oC for 5 min and incubating with T4 PNK (NEB, catalog no. M0201S) in a 10 μL reaction (7 μL precipitated RNA, 1 μL 10x T4 PNK Buffer, 1 μL SUPERase Inhibitor, 1 μL 10 U/μL T4 PNK) at 37oC for 1 hour. The reaction was stopped through heat inactivation at 65oC for 20 min. To ligate adaptor, samples were then incubated with 0.5 μL of 50 μM Universal miRNA Cloning Linker (NEB, catalog no. S1315S) and denatured at 65oC for 5 min. The denatured sample was then incubated with 1 μL T4 RNA Ligase 2, truncated KQ (NEB, M0373S), 1 μL 10x buffer, 6 μL 50% PEG 8000, and 1.5 μL water for 4.5 h at 25oC. Free adaptor was then removed by addition of 1 μL 10 U/μL 5’-Deadenylase (NEB, M0331S). 1 μL 10 U/μL RecJ Exonuclease (Lucigen/Epicentre, RJ411250). and 1 μL 20 U/μL SUPERase Inhibitor. Samples were then purified using Zymo RNA Clean & Concentrator-5 columns using the protocol above to preserve small RNAs (100 μL sample, 200 μL RNA binding buffer, 450 μL 100% ethanol). To perform reverse transcription, 10 μL sample was incubated with 2 μL of 1.25 μM RT primer (see table) and denatured at 65oC for 5 min. Reverse transcription (RT) was performed with primers containing sample barcodes and unique molecular identifiers. RT was performed with SuperScript III (Thermo Fisher, 18080-044) in a 20 μL reaction (48oC, 40 min). RNA was then hydrolyzed by adding 2.2 μL of 1N NaOH and incubating for 20 min at 98oC. Samples were then purified using Zymo RNA Clean & Concentrator-5 columns (100 μL sample, 200 μL RNA binding buffer, 450 μL 100% ethanol). RT products were size selected and gel extracted from 10% TBE Urea polyacrylamide gels as described above instead that DNA extraction buffer was used for overnight extraction (300 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA, 0.1% SDS). Eluate was then isopropanol precipitated overnight at -80oC overnight. Samples were then circularized with CircLigase (Illumina, CL4115K) in a 20 μL reaction (15 μL cDNA, 2 μL 10x CircLigase Buffer, 1 μL 1 mM ATP, 1 μL MnCl2, 1 μL CircLigase) for 12 hours at 60oC and subsequently purified by Zymo RNA Clean & Concentrator 5 columns (100 μL sample, 200 μL RNA binding buffer, 450 μL 100% ethanol) and eluted with 12 μL 10 mM Tris-HCl pH 8. 1 μL of library was used for PCR amplification with Phusion High-Fidelity DNA Polymerase (Thermo Fisher, catalog no. F530S) (98oC 30s, 98oC 10s, 65oC 10s, 72oC 5s) for 9-10 cycles (see table for primers). PCR product was PAGE purified from native 8% TBE polyacrylamide gels, extracted overnight using DNA extraction buffer, and isopropanol precipitated for > 2 h at -80oC. DNA was measured and quality controlled on the Agilent 2100 Bioanalyzer (High-Sensitivity DNA) by the Stanford Protein and Nucleic Acid (PAN) Facility. Libraries were sequenced by the Stanford Functional Genomics Facility (SFGF) on the Illumina NextSeq 500 (1x75nt).
Ribosome profiling, RNA-Seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

rRNA depleted total RNA

Data processing

For analysis pre-processing, UMI-tools63 was used to extract sample barcodes and UMIs. Reads were demultiplexed, sample barcodes were removed, and reads were quality filtered (fastq_quality_filter -Q33 -q 20 -p 70) using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
The 3’ adapter sequence CTGTAGGCACCATCAAT was removed using cutadapt64, and the 5’ end of each read was then removed using fastx_trimmer from FASTX-Toolkit. Raw files included have been preprocessed up to this point.
To remove reads that aligned to rRNA, tRNA, and snRNAs, reads were first aligned to these sequences using bowtie265 (bowtie2 parameters: -L 18) and subsequently discarded. Filtered reads that did not align to rRNA/tRNA/snRNAs were then aligned using bowtie2 (bowtie2 parameters: --norc -L 18) to an GRCm38/mm10 transcriptome reference derived from UCSC/GENCODE VM20 knownCanonical annotations filtered for high confidence transcripts that contain at least one of the following: a Uniprot ID, a RefSeq ID, or an Entrez ID.
PCR duplicates were then removed using UMI-tools. (Note 9 base UMI is in description of each read in FASTQ files)
Ribosome protected fragments (RPFs) were then parsed for uniquely aligned reads, separated into read length groups, and ribosome A site positions were determined by offsetting the distance of the 5’ end of each read to canonical start sites in each length group and adding 4 nucleotides62. RNA-Seq reads were also parsed for uniquely aligned reads and were assigned to the center of each read. RPFs and RNA-Seq reads were then counted using the transcriptome annotations detailed above.
Reads aligning to the CDS (with the first 15 codons and last 5 codons removed) were used for RPF libraries, and reads aligning to the entire transcript were used for RNA-Seq libraries.
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Counts aligning to CDS (with the first 15 codons and last 5 codons removed) for ribosome protected fragments; counts aligning to entire transcript for RNA-Seq

Submission date

Aug 12, 2019

Last update date

May 09, 2021

Contact name

Gerald Tiu

Organization name

Stanford University

Street address

279 Campus Dr., Beckman Center B309

City

Stanford