OTTR sequencing of DRT2 RNP + 16 uM dNTP reaction: alkaline hydrolysi... - SRA

SRX25615780: OTTR sequencing of DRT2 RNP + 16 uM dNTP reaction: alkaline hydrolysis treated
1 ILLUMINA (Illumina MiSeq) run: 150,410 spots, 45.1M bases, 27Mb downloads

Design: 10 L of 100 ng/L nucleic acids in water were heated to 95C for 2 min and immediately placed on ice. They were then mixed with 20 mM Tris-HCl pH 7.5, 200 mM KCl, 2 mM DTT, 5% PEG-8000, 2 mM ammonium sulfate, 0.7 mM sodium acetate, 2 mM MnCl2, 250 M ddATP, and 0.7 M BoMoC. After incubation at 30C for 90 min, 5 mM MgCl2 and 0.5 U of shrimp alkaline phosphatase (NEB) were added, incubated at 37C for 15 min, before addition of 5 mM EGTA and incubation at 65C for 5 min. Then, 0.5 mM MgCl2, 85 mM KCl, 200 M GTP, 40 M dTTP, 40 M dCTP, 40 M dATP, 90 nM RNA-DNA primer duplex (OTTR_c5_primer + OTTR_c5_RNA), 180 nM adapter template (OTTR_c3_AT), and 0.5 M BoMoC were added. The mixture was incubated at 37C for 20 min then 65C for 5 min. 1 L of the reaction was then directly used as template in a 50 L PCR with barcoded primers and PhusionFlash polymerase for 20 cycles and 30 seconds per elongation step. Adapter dimers were removed by pooling PCR products and purification with 0.6 volumes of SPRIselect beads (Beckman Coulter). Libraries were sequenced on a MiSeq in single-end mode (290 bp read 2).

Submitted by: Broad Institute

Study: Characterization of the DRT2 phage defense system from Klebsiella pneumoniae

PRJNA1144948 • SRP524491 • All experiments • All runs

show Abstracthide Abstract

To understand the mechanism of the defense-associated reverse transcriptase system DRT2 found in Klebsiella pneumoniae, we heterologously expressed it in E. coli and performed various forms of DNA sequencing with and without phage infection to look for the expressed cDNA. We also purified the DRT2 RNP and performed various biochemical assays, followed by DNA/RNA sequencing, to understand its composition in the resting state and during cDNA synthesis.

Sample: DNA/RNA extracted from purified DRT2 RNPs treated with 16 micromolar dNTPs

SAMN43047108 • SRS22262313 • All experiments • All runs

Organism: Escherichia coli BL21(DE3)

Library:

Name: DRT2RNP_16uMdNTP_OH_OTTR

Instrument: Illumina MiSeq

Strategy: RNA-Seq

Source: OTHER

Selection: RANDOM

Layout: SINGLE

Runs: 1 run, 150,410 spots, 45.1M bases, 27Mb

Run	# of Spots	# of Bases	Size	Published
SRR30147922	150,410	45.1M	27Mb	2024-08-06

ID:: 34623038

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