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Sample GSM7090248

Query DataSets for GSM7090248

Status

Public on May 30, 2023

Title

14_TRMB_B_S22

Sample type

SRA

Source name

delta-pyrFdelta-trmB cell culture

Organism

Haloarcula hispanica ATCC 33960

Characteristics

strain: AKS319
genotype: delta-pyrFdelta-trmB
cell type: delta-pyrFdelta-trmB cell culture
treatment: Hh-CA + uracil
time: 24hr
used in_de_analysis: TRUE
year-flowcell: 2022_S1

Growth protocol

Four single colonies of DF60 and ASK133 or AKS319 from 10-day old plates were inoculated in 5 mL YPC23 medium supplemented with 0.1% glucose and grown aerobically (250 rpm shaking) at 37C to stationary phase (optical density at 600nm ~ 4). Cells were washed twice with Hh-CA medium without glucose and transferred into fresh 50 mL Hh-CA with or without 0.1\% glucose at an initial optical density of ~ 0.4. After 24 hours cultures were collected and flash frozen with liquid nitrogen.

Extracted molecule

total RNA

Extraction protocol

After 24 hours storage in -80C, RNA was extracted from cell pellets using Absolutely RNA Miniprep kit (Agilent Technologies, Santa Clara, CA) followed by additional DNAse treatment with Turbo DNAse (Invitrogen). Total RNA was quantified using Agilent Bioanalyzer RNA Nano 6000 chip (Agilent Technologies, Santa Clara, CA). The absence of DNA contamination was determined on 200 ng samples of RNA in 25 cycles of PCR. Ribosomal RNA was removed from total RNA with the PanArchaea riboPOOL kit according to the manufacturer protocol (siTOOLs Biotech, Germany) and checked for depletion using Agilent Bioanalyzer RNA Nano 6000 chip as described as Described in Pastor et al. 2022.
rRNA-depleted RNA was used to construct the sequencing libraries with KAPA Stranded RNA-seq Library preparation (Kapa Biosystems) following manufacturer protocol. Fragment size of final libraries was measured using Agilent Bioanalyzer DNA 1000 chip and then pooled and sequenced on Illumina NovaSeq 6000 at the Center for Genomic and Computational Biology at Duke University (Durham, NC).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

66% of read pairs mapped to transcipts with appropriate mate pair orientation. 5,214 fragments mapped to pyrF (HAH_2085)

Data processing

Merged FASTQ files for samples that were loaded onto multiple lanes of a given flowcell, respecting read pair assignment. Ex) cat *_L001_R1_001.fastq.gz *_L002_R1_001.fastq.gz
Adapter sequences were removed with Trim_galore! 0.4.3, cutadapt 2.3, and and sample quality assessed with FastQC 0.11.7 using default parameters.
Reads were aligned to the Haloarcula hispanica ATCC33960 reference genome (GCF_000223905.1, accessed 2018-11-13) with Bowtie2 2.3.5.1 in paired-end mode: only pairs aligning concordantly in the --rf orientation were considered.
Aligned sequence files were converted to binary format, sorted, and indexed using samtools 1.10.
Read paris, or fragments, aligning to ORFs were counted using featureCounts 2.0.1 with arguments -p -B -s 2 and the Haloarcula hispanica ATCC33960 genome annotation (GCF_000223905.1, accessed 2022-10-19). Fragments were required to align concordantly in the correct strand orientation (reverse) to be counted.
In R, counts from 2021 and 2022 experiments were normalized relative to library size and batch corrected with DeSeq2 1.34.0. Pairwise correlations were computed using normalized counts for samples within each genotype+condition group, and samples with average R < 0.7 were considered outliers and were not used in downstream analyses. Only aAWT_B_noglu_24_S19_SP met these requirements for removal. Remaining samples were subjected to differential expression analysis.
Assembly: GCF_000223905.1
Supplementary files format and content: Text file of fragments mapping to genes. Each column is a sample, and rows are counts mapping to a given gene.

Submission date

Mar 09, 2023

Last update date

May 30, 2023

Contact name

Amy Schmid

E-mail(s)

[email protected]

Organization name

Duke University

Street address

3242 French Family Science Center