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| Status |
Public on Dec 09, 2022 |
| Title |
HB-Low-R1 |
| Sample type |
SRA |
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| Source name |
Hemangioblast Cell (HB)
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| Organism |
Mus musculus |
| Characteristics |
cell type: HM-1 ES dervied Hemangioblast cells
mouse strain: 129/ola
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| Growth protocol |
A genome-wide enhancer reporter assay was designed based on an enhancer reporter system designed by Wilkinson et al. (2013). Briefly the enhancer reporter functions by inserting a fragment of interest upstream of a HSP68 minimal promoter and Venus-YFP reporter by Gateway® cloning. The reporter construct is then transfected into the HM-1 ES cell line which has a non-functional HPRT locus. Using HPRT homology arms the report cassette becomes integrated into the HPRT locus by homologous recombination, also repairing the locus, enabling selection of clones with successful recombination. This enhancer reporter system was modified and optimised for genome-wide screening. To obtain genome-wide enhancer fragments for cloning into the reporter we isolated tn5 tagmented open-chromatin fragments based on the ATAC-Seq protocol 33 from cells of the five differentiation stages. Cells were obtained (2.5x105) from each differentiation stage (ES, HB, HE1, HE2, HP) as detailed in the serum IVD method. Cells were pelleted in 5 aliquots of 50x103 cells and following the ATAC-Seq protocol each cell pellet had a transposition mix added (2x TD Buffer 25µl, TN5 Transposase 2.5 µl, PBS 16.5 µl, 1% Digitonin 0.5 µl, 10 % Tween-20 0.5 µl, H2O 5 µl) and cells were gently resuspended by pipetting. The transposition reaction was then incubated in a shaker at 700 RPM, 37°C for 30 minutes. The 5 reactions were then combined and DNA purified using the Qiagen MinElute® Reaction Clean-up kit and eluted in 26.5 µl H2O. One fifth of the purified reaction from each stage was used to produce ATAC-Seq libraries for direct sequencing and the remainder had linkers added incorporating AttB Gateway® cloning sites to enable insertion into a Gateway® donor vector (pDONR 221). The linkers were designed based on the tn5 transposase sequence and added by PCR in 2 steps. Initially 5 cycles of PCR were performed (Transposed DNA 20 µl, H2O 20 µl, 25 µM AttB tn5 Fwd primer 5 µl, 25 µM AttB tn5 Rev primer 5 µl, NEBNext Master Mix 50 µl) with the following conditions, 72°C for 5 minutes, 98°C for 30 seconds, 5 cycles of 98°C 10 seconds, 63°C 30 seconds and 72°C 1 minute and a final hold at 4°C. To optimize the required cycle number, the material from the initial PCR reaction was split and a 5 µl was used for a separate qPCR reaction (0.125 µM AttB tn5 Fwd Primer, 0.125 µM AttB tn5 Rev Primer, 1 x SYBR Green I, NEBNext Master Mix) with the following conditions: 72°C for 5 minutes, 98°C for 30 seconds, 35 cycles of 98°C 10 seconds, 63°C 30 seconds and 72°C 1 minute using a StepOnePlus™ Real-Time PCR System (Thermo Fisher). Using the raw data from the cyan channel the number of cycles for 25% amplification was calculated. The number of cycles obtained from the qPCR side reaction was then used to amplify the remainder of the material as follows, 98°C for 30 seconds, cycles of 98°C 10 seconds, 63°C 30 seconds and 72°C 1 minute with a final hold at 4°C. The resulting material was size selected, to optimise for material originating from enhancers, for fragments between 350 and 650 bp by electrophoresis on a 1.2% agarose gel with ethidium bromide and gel extraction was performed using the Qiagen MinElute® Gel Extraction Kit following manufactures protocol. To generate attL-flanked entry clones compatible with cloning into the reporter vector the size selected material was cloned into the pDONR™221 (Thermo Fisher) by BP Gateway® reaction (100 fmol pDONR221, 100 fmol PCR material, 2 µl BP Clonase II, made up to 20µl with TE buffer (pH 8.0) the reaction was incubated for 18 hours at 25°C and ended by treatment with 0.2µg proteinase K and incubation at 37°C for 10 minutes. NEB 10-beta Electrocompetent cells were transformed with the plasmid by electroporation with a Bio-Rad GenePulser using program EC1 (2.0 kV, 200 Ohm, 25 µF), after addition of outgrowth media and incubation at 37°C, 250 rpm for 1 hour the bacteria were spread onto 150 mm agar plates containing 50 µg/ml kanamycin. Following overnight incubation at 37°C, 10 ml of LB was added to each plate and the bacterial lawn scraped off using a cell scraper. Plasmid DNA was extracted from the collected bacteria by Maxi-prep using the NucleoBond Xtra Midi EF kit following manufacturer’s instructions. The resulting product was eluted in 500 µl H2O. The chromatin fragments were then transferred into the enhancer reporter cassette in the pSKB-GW-Hsp68-Venus vector 1 by Gateway® LR reaction (150ng Entry clone (pDONR), 150 ng pSKB, 2 µl LR Clonase II, made up to 10µl with TE buffer (pH 8.0) ) the reaction was incubated for 1 hour at 25°C and ended by treatment with 0.2 µg proteinase K and incubation at 37°C for 10 minutes. As with the BP reaction product, NEB 10-beta Electrocompetent cells were transformed with the material and after overnight incubation on agar plates containing 200 µg/ml ampicillin the bacteria were collected and the plasmid library extracted by Maxi-Prep. To obtain the highest possible diversity of fragments the cloning was carried out 10 times for material from each differentiation stage with the pDONR 221 library material being combined before moving to the LR clonase reaction. The HM-1 ES cell line was cultured on gelatinised culture plates in ES-Media (DMEM, 15% FCS, 1 mM Sodium pyruvate, 1x Pen/Strep, 1x L-glutamine, 0.15 mM MTG, 25 mM Hepes buffer and 1u/ml ESGRO LIF (Sigma-Aldrich)) at 37 °C, 10% CO2 and split every 48 hours to maintain healthy cells. Prior to transfection the cells were treated for 1 week with 1 x 6-TG to remove any cells with a functional HPRT locus. The plasmid libraries were mixed and digested with PmeI (NEB) to linearise the plasmids prior to transfection. To obtain optimum homologous recombination efficiency CRISPR guides were designed flanking the region and cloned into the PX458 Cas9 and sgRNA expression vector. For each experiment, 500x106 cells were transfected using a Nucleofector®-4D (Lonza) with the P3 Primary Cell X kit with 5x106 per cuvette 10 ug pSKB enhancer reporter plasmid and 10 ug of PX458-HPRT CRISPR plasmid using program CG-104. Following electroporation, the cells were plated on gelatinised plates in ES-media with the addition of 50 µM SCR7 pyrazine (Sigma) to inhibit Non-homologous end joining, promoting homologous recombination following cutting by Cas9. After 12 hours the media was changed and 1 x HAT (Hypoxanthine-Aminopterin-Thymidine (Sigma)) was added to select for clones with successful recombination. The cells were then maintained in media containing HAT for one week and HT (hypoxanthine and thymidine (Sigma)) for a further 2 passages to prevent the cells from dying after the withdrawal of HAT, whereby all cells were kept and replated after splitting. The selected cells were then put into IVD cultures and differentiated into HB, HE1, HE2 and HP (as described in Obier et al (2016)) and sorted by FACS. In addition to the previously mentioned gating for obtaining the cell populations the cells were also sorted into negative, low, medium and high Venus-YFP populations representing the activity of the enhancer fragment driven minimal promoter. The YFP-Venus negative population was gated based on a cell line containing a reporter construct with only the minimal promoter and reporter with no enhancer fragment.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from the sorted cell populations using the Qiagen DNA Micro kit following manufacturer’s instructions.
To amplify the fragments contained in the reporter cassettes and add sequencing adaptors a PCR using indexed primers compatible with Illumina sequencing and targeted against the tn5 transposase sequence ((0.5 µM Nextera PCR Primer i5, 0.5 µM Nextera PCR Primer i7, 25 µl NEBNext Master Mix). Dependant on the amount of DNA obtained from the sorted cells between 25 and 30 PCR cycles were performed with the following conditions 98°C for 30 seconds, cycles of 98°C 10 seconds, 63°C 30 seconds and 72°C 1 minute with a final hold at 4°C. Libraries were quantified using a Bioanalyzer 2100 (Agilent) with a High Sensitivity DNA Chip and pooled at equal concentration for sequencing. The final pool concentration and size was then confirmed by Kappa Library Quantification Kit (Roche) and Bioanalyzer. The pooled libraries were sequenced on a Next-Seq 550 as a paired end run with a 150 cycle High Output kit by the Genomics Birmingham Sequencing Facility.
Enhancer Reporter Sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paired end sequencing reads were trimmed using Trim Galore (Krueger F, 2021) with the parameters --nextera --length 70 –paired.
The reads were then aligned to the mm10 genome using bowtie2 using parameters --very-sensitive --fr --no-discordant -X 600 --no-mixed.
Aligned reads were filtered for only those which were properly paired and with a mapq score of over 40 using Samtools and output as a bedpe file.
This was further converted to a bed file of enhancer fragments by taking the first co-ordinate of read 1 and the final co-ordinate of read 2 for each pair. Duplicate fragments were removed using the uniq function and fragments were further filtered using the intersect function on Bedtools for any which had 100% overlap with an off target background fragment list which was created by producing a library from un-transfected cells.
The fragments were then filtered using Bedtools intersect for those which were found in open chromatin, defined by DNaseI-Seq, in Goode et al (2016) and further by a corresponding ATAC peak in the correct differentiation stage in HM-1 cells.
Finally, the enhancer fragments were annotated using the annotatePeaks function of Homer for those within 1.5 kb of a TSS (promoter fragments) and distal fragments representing genuine enhancers.
Assembly: mm10
Supplementary files format and content: Bed files of filtered enhancer fragment co-ordinates
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| Submission date |
Mar 16, 2022 |
| Last update date |
Dec 09, 2022 |
| Contact name |
Peter Keane |
| E-mail(s) |
p.keane@bham.ac.uk
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| Organization name |
University of Birmingham
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| Department |
Institute for Cancer and Genomic Sciences
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| Street address |
Vincent Drive
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| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE198773 |
A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification (Enhancer Reporter Sequencing) |
| GSE198775 |
A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification |
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| Relations |
| BioSample |
SAMN26725018 |
| SRA |
SRX14475327 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5956772_HB_L_1_enhancer_fragments.bed.gz |
66.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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