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| Status |
Public on Dec 01, 2023 |
| Title |
trmB_a_noglu |
| Sample type |
SRA |
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| Source name |
H26 ∆pyrE2∆trmB
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| Organism |
Haloferax volcanii DS2 |
| Characteristics |
strain: AKS154
genotype: H26 {delta}pyrE2{delta}trmB
cell type: archaeal cell culture
treatment: HvCA + uracil
time: 4hr
year-flowcell: 2021_S1
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| 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.
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| 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). For all samples, the absence of DNA contamination was determined on 200 ng samples of RNA in 25 cycles of PCR and total RNA was quantified using Agilent Bioanalyzer RNA Nano 6000 chip (Agilent Technologies, Santa Clara, CA). For 2019 samples, ribosomal RNA was removed with Ribo-Zero rRNA Removal Kit (now discontinued, Illumina, San Diego, CA). For 2021 samples, extraction was followed by additional DNAse treatment with Turbo DNAse (Invitrogen), and 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.
For 2019 samples, 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 HiSeq4000 at the Duke Sequencing and Genomic Technologies Core Facility at Duke University (Durham, NC). For 2021 samples, sequencing libraries were constructed with NEBNext UltraII Directional RNA Library Preparation Kit (New England BioLabs, #E7760). The fragment size of all libraries was measured using Agilent Bioanalyzer DNA 1000 chip and then pooled and sequenced on NovaSeq6000 at the Center for Genomic and Computational Biology at Duke University (Durham, NC).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
trmB_a_noglu_S32
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| 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 Haloferax volcanii DS2 reference genome (GCA_000025685.1, accessed 2023-09-11) with Bowtie2 2.3.5.1. Alignment by Bowtie2 required stand-specific read orientation for libraries constructed in the 2021 experiment. 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.
Reads (for samples from 2019) or read paris (2021 samples) aligning to ORFs were counted using featureCounts 2.0.1 with arguments -p -B -s 2 and the DS2 reference genome annotation (GCA_000025685.1, accessed 2023-09-11). For 2021 samples, fragments were required to align concordantly in the correct strand orientation (reverse) to be tallied.
In R, counts from 2019 and 2021 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.
Assembly: GCA_000025685.1
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.
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| Submission date |
Nov 24, 2023 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Amy Schmid |
| E-mail(s) |
amy.schmid@duke.edu
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| Organization name |
Duke University
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| Street address |
3242 French Family Science Center
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
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| Platform ID |
GPL30888 |
| Series (2) |
| GSE248564 |
Transciptome profile of TrmB and TbsP in Haloferax volcanii [RNA-seq] |
| GSE248566 |
Transciptome profile of TrmB and TbsP, Genome-wide binding of TbsP (HVO_2861) in Haloferax volcanii |
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| Relations |
| BioSample |
SAMN38412635 |
| SRA |
SRX22635653 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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