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| Status |
Public on Oct 08, 2019 |
| Title |
AH_HB2 |
| Sample type |
SRA |
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| Source name |
EMSA on Synthetic oligo nucleotides, Hta-bound slow band
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| Organism |
synthetic construct |
| Characteristics |
strain: n/a
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| Treatment protocol |
T. acidophilum cells self-lyse at pH>6 (Robb et al. 1995). Pellets were therefore directly re-suspended in ice-cold MNase digestion buffer (10mM Tris, 5mM Ca2+, pH8) and homogenized by 20 passages through a Dounce homogenizer, on ice. Unfixed crude lysates were digested in the presence of 4U/mL of MNase (Thermo Fisher) at 37°C. Digestion was stopped by addition of EDTA to a final concentration of 20mM. Samples were incubated for an additional 30min at 37°C in the presence of RNAse A to a final concentration of 0.5mg/mL and then overnight at 65°C in the presence of SDS (1%) and proteinase K (125µg/mL). Undigested lysate was incubated as above but without addition of enzyme. Undigested DNA was then sonicated using a Covaris S220 sonicator with the following settings: peak power: 175, duty: 10, cycle/burst: 200 for 430s (target size: 150bp).
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| Growth protocol |
T. acidophilum strain 122-1B2 was obtained from DSMZ (https://www.dsmz.de/) and cultured using the medium described in (Searcy and Stein 1980), supplemented to a final concentration of 2g/L yeast extract (BD Biosciences). The medium was boiled for five minutes and allowed to cool to 58°C before inoculation. Cultures were incubated at 59°C with shaking (90rpm) in an INFORS Thermotron incubator.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HB Corresponds to HTa bound Slower Samples. For EMSA, fresh HTa aliquots were mixed with a pool of randomized oligos -500ng per reaction- in a mass ratio of 0.2 in a buffer containing a final concentration of 1% glycerol, 20 mM NaCl, 30 mM Tris-Hcl, 0.7 mM EDTA in a final volume of 24µL. 4µL of 6x loading buffer (without SDS, NEB #B7025S) were added just before gel electrophoresis. For each replicate 10 reaction were run on the same gel (12 % polyacrylamide) at 90V for 70 min at 4°C, in a prechilled 10 mM Tris pH8 buffer. DNA was stained with EtBr and gel washed twice before imaging. After imaging, DNA the wo visible bandshifts were separately extracted from the gel following the crush ad soak method, pooling the ten wells for each replicate. DNA concentration was measured by Qbit before library preparation.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
EMSA-seq assay
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| Data processing |
Library strategy: EMSA-seq
Basecalls performed using CASAVA 1.8.4
Reads were aligned to NC_002578.1 T.acidophilum genome assembly or to E.Coli C600 genome (CP031214) obtained from ncbi, using Bowtie2
Paired-end 100bp reads were first trimmed using BBDuk v37.64 (parameters: ktrim=r, k=21, hdist=1, edist=0, mink=11, qtrim=rl, trimq=30, minlength=10, ordered=t, qin=33) and then merged using BBmerge v37.64 (parameters: mininsert=10, mininsert0=10).
The merged reads were mapped to the T. acidophilum DSM1728 genome (GCA_000195915.1) or to E.Coli C600 genome (CP031214) using Bowtie2 (-U).
Coverage tracks were computed using deeptools bamCoverage (RPGC normalization, effective genome size: 1564906bp), measuring coverage for reads of sizes 40-65bp and 70-100bp separately. Reads from the sonicated DNA control samples were mapped in paired-end mode. Coverage was computed independently of read size and smoothed over 10kbp to avoid introducing noise from the input into the MNase signal. Genome-wide coverage of MNase-digested samples was then divided by its corresponding undigested DNA sample to remove bias in coverage associated with replication. Lastly, coverage was converted into a Z-score using the scale function in R.
Genome_build: T.acidophilum : NC_002578.1 ; E.coli : CP031214
Supplementary_files_format_and_content: Bigwig
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| Submission date |
Oct 08, 2019 |
| Last update date |
Oct 09, 2019 |
| Contact name |
Tobias Warnecke |
| E-mail(s) |
molecular.systems.laboratory@gmail.com
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| Organization name |
Imperial College
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| Street address |
Du Cane Road
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| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
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| Platform ID |
GPL19604 |
| Series (2) |
| GSE127728 |
The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog |
| GSE138576 |
The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog (Mnase-seq and EMSA-seq) |
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| Relations |
| BioSample |
SAMN12993513 |
| SRA |
SRX6964061 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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