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Sample GSM4522992

Query DataSets for GSM4522992

Status

Public on Aug 04, 2020

Title

Heart_tRNA_1_DM

Sample type

SRA

Source name

Heart

Organism

Mus musculus

Characteristics

cell type: NA
passages: NA

Treatment protocol

Growth protocol

Extracted molecule

total RNA

Extraction protocol

All mouse tissue samples were isolated from C57B/6J mice. Samples were received on dry ice, and stored as whole tissue at -80°C. Samples were thawed on ice and 1mL of Trizol was added per 100g of dissected whole tissue. On ice, samples were masticated and passed through successively higher gauges of needles to ensure a homogeneous mixture. Samples were stored at -80°C until further processing. RNA was isolated according to manufacturer’s suggested conditions with the addition of a second ethanol precipitation step with two 75% ethanol washes following isopropanol precipitation. Samples were resuspended in distilled H2O and stored at -80°C until library preparation.
Total RNA samples were quantified using a nanodrop spectrophotometer (ThermoFisher) prior to library preparation and RNA integrity was checked on a 1.2% denaturing formaldehyde agarose gel. To remove 3’ conjugated amino acids, total RNA was deacylated in deacylation buffer (final concentration 20mM Tris-HCL pH=9.0) at a final concentration of 1ug/uL. 1 ug of deacylated total RNA from each sample was subject to demethylase treatment (rtStarTM tRNA-optimized First-Strand cDNA Synthesis Kit, ArrayStar) followed by library preparation. 10 pmol of the 3’ and 10 pmol of the 5’ single-stranded adapter mix (2.5 pmol of each adapter 5’-TGrGrA-3’, 5’-TGrGrT-3’, 5’-TGrGrG-3’, 5’-TGrGrC-3’; Table 1) were added to a 200uL thin-walled amplification tube and denatured at 95°C for 2 minutes. Then annealing buffer was added to a final concentration of 5mM Tris-HCl (pH 8.0), 0.5mM ethylenediaminetetraacetic acid (EDTA) and 10mM MgCl2 and incubated at 37°C for 15 minutes hybridizing the annealed double-stranded adapter to the total RNA. The ligation reaction was catalyzed by 5U/uL of RNA ligase 2 (NEB) with manufacturers suggested conditions at 37°C for 60 minutes then 4°C at 60 minutes. All reactions were ethanol precipitated with glycoblue (Thermo Fisher) with two 75% ethanol washes, then suspended in 10ul of dH2O. Following ligation, synthesis of cDNA began with hybridization of the reverse transcriptase primer to the ligated total RNA with a final concentration of 0.5pmol/uL. The samples were incubated at 70°C for 2 minutes and temperature was reduced to 37°C by 0.1°C/sec. Synthesis of cDNA was achieved using Superscript IV at 55°C for 60 minutes. To remove DNA-RNA dimers following cDNA synthesis, we hydrolyzed RNA with a final concentration of 0.1N NaOH in dH2O at 98°C for 20 minutes. All reactions were ethanol precipitated with glycoblue (ThermoFisher) with two 75% ethanol washes, then suspended in 12ul of dH2O. cDNA libraries were separated using 7M urea 6% denaturing polyacrylamide gels. Gels were stained with 1X SYBR gold (Thermo Fisher) in 1X TBE for 15 minutes and regions of interest (60-200 nucleotides) were excised on a UV light box. Gel slices were sheared through the bottom of a 0.5 mL tube nested in a 1.7 mL tube by centrifugation then suspended in 400uL of DNA elution buffer (300mM NaCl, 10mM Tris (pH=8.0, 1mM EDTA), incubated on dry ice for 30 minutes, and allowed to incubate at room temperature overnight on a standing rotator. Libraries were isopropanol precipitated with glycoblue followed by two 75% ethanol washes. Linear cDNA libraries were then suspended in 12ul of dH2O. Circularization of cDNA libraries was performed with CircLigase (Epicentre) with 0.5U/uL using manufacturer’s suggested conditions at 37°C for 1 hour. The reaction was terminated with incubation at 80°C for 20 minutes. All reactions were ethanol precipitated with glycoblue with two 75% ethanol washes, then suspended in 12.5ul of dH2O. cDNA libraries were amplified using the NEBnext Ultra II Q5 next-generation master mix (NEB) with manufacturer’s suggested conditions. HEK293 libraries were amplified for 7 cycles and mouse tissue libraries amplified for 7-9 cycles. Amplified libraries were gel purified from 2% agarose gels stained with 0.05mg/ml Ethidium Bromide. Regions of interest (100-250bp) were excised on a UV light box and purified using the Qiaquick (Qiagen) gel extraction kit using all optional steps. All libraries were ethanol precipitated with glycoblue (thermo) with two 75% ethanol washes, then suspended in 10ul of dH2O before sequencing.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 550

Description

Mouse_tissue_variant_counts_DM

Data processing

Processing: cutadapt ver 1.12 was used twice in succession with the following parameters first to remove two degenerate bases encoded in the RT primer (--cut 2), potential 5' adapter (-g TCCAACTGGATACTGGN -e 0.2) and then to remove the 3' CCA + adapter (-a CCAGTATCCAGTTGGAATT -e 0.2).
Mapping: bowtie2 ver 2.3.5 was used to map processed reads to a reference generated from high confidence tRNAs (unique sequences collapsed) defined in GtRNAdb (mm10) using the following parameters: (--quiet --min-score G,1,8 --local -D 20 -R 3 -N 1 -L 10 -i S,1,0.5).
Downstream analyses: Mapped sam files were converted to bam files using samtools ver 1.9 (samtools view). Further analyses were carried out in Rstudio ver 1.1.463 (R ver 3.5.2 (2018-12-20) -- "Eggshell Igloo"). Read count tables over tRNA genes were generated by using featureCounts from the Rsubread package using the following parameters: strandSpecific = 1,useMetaFeatures=F,minMQS = 10.
mm10; gtRNAdb Release 18
CSV files of variant counts and coverage for every base at the isodecoder level from mouse tissues.

Submission date

May 07, 2020

Last update date

Aug 05, 2020

Contact name

Jeff Coller

E-mail(s)

[email protected]

Phone

216-368-0299

Organization name

Case Western Reserve University