|
| Status |
Public on Aug 25, 2021 |
| Title |
HpyA_exp_opt_IP2 |
| Sample type |
SRA |
| |
|
| Source name |
archaeal cells_Δura3ΔhpyA/hpyA-HA_Exponential
|
| Organism |
Halobacterium salinarum |
| Characteristics |
strain: KAD128
genotype: {delta}ura3{delta}hpyA/hpyA-HA
growthphase: Exponential
epitope tag: HA
cell type: archaeal cells
antibody: Anti-HA(ab9110)
|
| Growth protocol |
Colonies of AKS134 (Empty vector negative control) and KAD128 (HpyA-HA; 4 biological replicates) were pre-cultured in rich media for ~120 hrs and diluted to OD~0.02. Paired replicates were grown in optimal and reduced salt rich media (4.2M and 3.4M NaCl respectively) supplemented with Mevinolin 0.1ug/mL for plasmid maintenance. At exponential phase (OD600 ~0.2-0.35;36-50 hrs) and stationary phase, (OD600~1.4-1.7, ~70-140 hrs.), 45 mL cell pellets were harvested, crosslinked with formaledhyde, and frozen for immunoprecipitation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crrosslinked and immunoprecipitated with HA antibody (ab9110) as described in Wilbanks et al 2012 (doi: 10.1093/nar/gks063) with the following changes to the protocol: the cross-linking reaction was allowed to proceed for 20 minutes rather than 10, and cell pellets were resupended in 800 μL lysis buffer instead of 1.6mL.
Libraries were constructed using the KAPA Hyper Prep kit and Illumina TruSeq adapters according to manufactuer's instructions. DNA library quality was assessed by Bioanalyzer using a High Sensitivity DNA chip (Agilent) . Samples were pooled and run on an Illumina HiSeq 4000 (Duke Sequencing and Genomics Technologies core) for single-end sequencing. 1st batch contained all stationary phase samples and 2nd batch contained all exponential phase samples.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Gzipped FASTQ files were assessed for quality using FastQC and adapter sequences trimmed using Cutadapt/Trim Galore.
Trimmed fastq files were aligned to the Halobacterium salinarum NRC-1 genome (GCF_000006805.1_ASM680v1) using Bowtie2. Samtools was used to generate and sort BAM files. Sorted BAM files were used for further analysis.
Peak-calling was done using MACS2 software on individual samples (qval cutoff 0.01, nomodel parameter used) and narrowPeak files with peak locations, enrichment, and q-values were generated.
WIG files were generated using the samtools mpileup command. Per-base count files were generated using bedtools genomecov.
Genome_build: GCF_000006805.1_ASM680v1
Supplementary_files_format_and_content: WIG file
|
| |
|
| Submission date |
Aug 20, 2021 |
| Last update date |
Aug 26, 2021 |
| Contact name |
Amy Schmid |
| E-mail(s) |
amy.schmid@duke.edu
|
| Organization name |
Duke University
|
| Street address |
3242 French Family Science Center
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL28343 |
| Series (2) |
| GSE182492 |
Binding profile of Halobacterium salinarum histone [ChIP-Seq] |
| GSE182514 |
Binding and Transcriptional profile of Halobacterium salinarum histone |
|
| Relations |
| BioSample |
SAMN18009712 |
| SRA |
SRX10138035 |
