ssDNA sequencing of DRT2-expressing E. coli: uninfected cells, YCAA m... - SRA

SRX25615788: ssDNA sequencing of DRT2-expressing E. coli: uninfected cells, YCAA mutant, S1-nuclease treated
1 ILLUMINA (Illumina MiSeq) run: 14,714 spots, 4.4M bases, 2.8Mb downloads

Design: DNA (500 ng) was treated with and without 100 U S1 Nuclease (Thermo Scientific) in 1 reaction buffer at 25C for 2h before dilution with 50 mM Tris-HCl pH 7.5, extraction with phenol-chloroform-isoamylalcohol (25:24:1), and ethanol precipitation. Half of the precipitated DNA was used as input for strand-specific DNA libraries. Input DNA was supplemented with 1 fmol of 200 nt control ssDNA (ssd_spike) and incubated with 20 U terminal transferase (NEB) and 4 M dATP in 1 reaction buffer (without CoCl2) at 37C for 30 min then 70C for 5 min. 2.5 pmol of primer ssExt_pT9_anchor was then annealed to the DNA, followed by addition of 1 mM dNTPs and 5 U Klenow Fragment (exo-, NEB). The reaction was incubated at 37C for 30 min then 65C for 5 min, and products were purified using the QIAquick PCR purification kit (Qiagen). Adapters ssd_adapt_top and ssd_adapt_bottom were annealed, and 100 fmol was ligated to 13% of the recovered DNA using Blunt/TA Ligase Master Mix (NEB). Ligation reactions were amplified with PhusionFlash PCR master mix and barcoded primers for 20 cycles. Amplicons larger than the 147 bp adapter dimer were purified by gel extraction and 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

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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 from E. coli expressing DRT2 YCAA mutant, S1 nuclease treated

SAMN43047102 • SRS22262319 • All experiments • All runs

Organism: Escherichia coli

Library:

Name: YCAA_noT5_S1_ssDNA

Instrument: Illumina MiSeq

Strategy: WGS

Source: GENOMIC

Selection: RANDOM

Layout: PAIRED

Runs: 1 run, 14,714 spots, 4.4M bases, 2.8Mb

Run	# of Spots	# of Bases	Size	Published
SRR30147932	14,714	4.4M	2.8Mb	2024-08-06

ID:: 34623046

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