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Sample GSM7054879

Query DataSets for GSM7054879

Status

Public on Sep 27, 2023

Title

T7 E. coli tRNAs control treatment (minus DMS) 10mers / 1: 1 / 0.5 replicate 3

Sample type

SRA

Source name

T7 transcripts of E. coli tRNAs

Organism

synthetic construct

Characteristics

cell line: n/a
treatment: control

Extracted molecule

total RNA

Extraction protocol

Trizol extraction
To facilitate ligation onto the 3’-end of RNAs, the ligation adapter must be 5’-end adenylated. The adapter adenylation reaction was prepared as follows: water, 10 μM Mth RNA ligase (M2611A), 200 μM ATP, 20 μM of the ligation adapter (called 3' adapter in supplemental material excel sheet), and 1X 5’ DNA Adenylation reaction buffer (B2610S). The reaction was incubated at 65 °C for 1 h, after which, the enzyme was heat inactivated at 85 C for 5 min. We performed the RT primer ligation reaction as follows: 25% PEG 8000, 1X T4 RNA Ligase Reaction Buffer, 3 μM adenylated DNA oligonucleotide from the previous step, 4 μL of RNA recovered from a DMS experiment, and 200 U of T4 RNA Ligase 2 truncated (New England Biolabs). The reaction was incubated at 16 °C overnight, and the next day, the ligated product was fractionated alongside an unligated starting material lane on a 10% denaturing (8.3 M urea 1X TBE) polyacrylamide gel. The gel was stained with SYBR-GOLD and bands were visualized with UV light. The ligated band was excised, cut into ~1 mm x 1mm squares, and placed in a 1.7 mL tube with 450 μL of TEN-250 buffer and crush and soaked at 4 °C overnight. The ligated product was recovered by ethanol precipitation. The ligated products were reverse transcribed by marathon reverse transcriptase in the presence of Mn2+ ions to induce mutations at DMS modification sites and encourage readthrough.9,10; The ligated product and reverse transcription primer were renatured together at 90 °C for 1 min and then cooled to 4 °C for 10 min to hybridize them. The marathon reverse transcription reaction contained the following: 50 mM Tris-HCl (pH 8.3), 200 mM KCl, 5 mM dithiothreitol, 20% glycerol, 2 mM MnCl2, 250 μM of each dNTP, less than 500ng of ligated product, 250 nM of reverse transcription primer (called RT primer in supplemental material excel sheet) and 10-20 U of Marathon Reverse Transcriptase (Kerafast). The reaction was incubated at 42 °C for 1 h, then ~200 mM NaOH was added, and the reaction was heated to 95 °C for 5 min to denature the reverse transcriptase and degrade the RNA template. Then an equal volume of 2X formamide loading dye (0.025% w/v bromophenol blue, 90% formamide, 10 mM EDTA) was added and the sample was fractionated on a 10% denaturing (8.3 M urea 1X TBE) polyacrylamide gel. The gel was stained with SYBR-GOLD, bands were visualized under UV-light, the RT product band was excised, cut into ~1 mm x 1mm squares, placed in a 1.7 mL tube with TEN-250 buffer and crush and soaked at 4 °C overnight. The reverse transcription product was recovered by ethanol precipitation. The single-stranded cDNA from the reverse transcription was circularized using CircLigase II. The reaction was prepared as follows: 1X Circligase reaction buffer (Lucigen), 2.5 mM MnCl2, 1 M betaine, single-stranded cDNA from the reverse transcription, and 3.3 U of CircLigase II (Lucigen). The reaction was incubated at 60 °C for 2 h. The circularized product was used in indexing PCR without further purification. The PCR was prepared as follows, 1X Phusion HF reaction buffer, 0.5 μM indexing primer, 0.5 μM reverse primer (called PCR reverse in supplemental material excel sheet), 200 μM of each dNTP, circularized DNA, and 0.5 U of Phusion HF DNA Polymerase. The thermocycler program was based on that from Yamagami et al.2 and was performed as follows: initial denaturation at 98 °C for 30 sec, denature at 98 °C for 10 sec, anneal at 60 °C for 10 sec, elongate at 72 °C for 5 sec, repeat steps 2-4 for 17 cycles before a final extension at 72 °C for 5 min. PCR products were fractionated on an 8% non-denaturing TBE polyacrylamide gel ran at 180 V for 90-120 min. The gel was stained with SYBR-GOLD, bands were visualized with UV light, the PCR product band was excised, cut into ~1 mm x 1mm squares, and placed in a 1.7 mL with TEN-250 buffer and crush and soaked at 4 °C overnight. The indexed library was recovered by Nucleospin Gel and PCR Clean-Up kit (Macherey-Nagel). Afterwards, the concentrations of each library were measured via a micro-volume UV-vis spectrophotometer (Denovix). Before the libraries were sequenced, they were evaluated for quality and appropriate length by the TapeStation system (Agilent). To determine the concentration of each library, qPCR was performed. Libraries were sequenced by Illumina NextSeq (150 nt single-end sequencing and High-output flow cells) with the standard Illumina primer.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 2000

Description

Library 54
T7_10mers_11_05_merged_out.tar.gz

Data processing

The adapters on the 3'-end of the reads were removed by cutadapt version 2.6 with Python version 3.6.13
ShapeMapper2 version 2.1.5 was used to align the reads to the sequences of the tRNA genes retrieved from the genomic tRNA database.
ShapeMapper2 version 2.1.5 was used to calculate the normalized DMS reactivity with the min-mutation-separation set to 1 for structure prediction and 0 for natural mutational profiling. The DMS reactivity was divided by the average DMS reacvtivity of the top 10% of the reactive nucleotides.
Then the correlation of the DMS reactivity between replicate experiments was evaulated by a custom R script available at https://github.com/MacMeyer1/Scripts-to-make-figures-for-tRNAs-in-peptide-rich-droplets
If the DMS reactivity between replicates was well correlated, the raw fastq files containing the reads were combined and ShapeMapper2 version 2.1.5 was used to again align the reads and calculate the normalized DMS reactivity.
Supplementary files format and content: Each processed file is a tar.gz directory containing the output of ShapeMapper2 version 2.1.5.
Library strategy: DMS-Map-Seq

Submission date

Feb 21, 2023

Last update date

Sep 27, 2023

Contact name

McCauley O'Brien Meyer

E-mail(s)

mcobmeyer35@gmail.com, mxm1357@psu.edu

Organization name

Penn State University

Department

Biochemistry and Molecular Biology