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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 26, 2022 |
| Title |
CRISPRaX_Screen_Top_r1 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14-STN-dTsixP
condition: Top
replicate: rep1
strain background: 129P2/Ola
assay: CRISPRa-Screen
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| Treatment protocol |
E14-STN-dTsixP cells were passaged for two passages in Serum+LIF-containing medium before a total of 1.200.000 cells were transduced with viral supernatant of the sgRNA library (MOI = 0.3). Puromycin selection (1 ng/µl, Sigma) was started 48 h after transduction and kept until the end of the experiment. Four days after transduction, 7.200.000 cells were differentiated by LIF withdrawal for 2 days. Expression of CRISPRa-SunTag system was induced using doxycycline (Clontech, 1 µg/ml) one day before differentiation and kept throughout the rest of the protocol. Cells were harvested with trypsin to reach a single cell suspension for Flow-FISH after 2 days of differentiation.
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| Growth protocol |
E14-STN-dTsixP cells were grown on gelatin-coated flasks in serum-containing ES cell medium (DMEM (Sigma), 15% ESC-grade FBS (Gibco), 0.1mM β-mercaptoethanol, 1000 U/ml leukemia inhibitory factor (LIF, Millipore)) at a density of 3.13*10^4 cells/cm2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The PrimeFlow RNA assay (Thermofisher) was used according to the manufacturer’s recommendations. Specifically, the assay was performed in conical 96-well plates for a total of 200 million cells per replicate with 5 million cells per well using Xist specific probes, labelled with Alexa-Fluor647 (VB1-14258). 20 million cells were snap-frozen after the two fixation steps to be used as the Unsorted fraction. Samples were resuspended in PrimeFlow™ RNA Storage Buffer before flow cytometry. Here, four different populations were sorted according to Xist expression. In more detail, a Xist-negative population was taken from the bottom-15% of Xist-expressing cells. Three Xist-positive populations were taken in 15%-intervals from the top Xist-expressing cells (0-15% = High, 15-30% = Medium, 30-45% = Low). Around 11-15 million cells were recovered per fraction. After sorting, the cell pellets were snap-frozen and stored at -80°C. DNA from frozen cell pellets was isolated through phenol/chloroform extraction. Cell pellets were thawed and resuspended in 250 µl of lysis buffer (1% SDS (Thermo Fisher Scientific), 0.2 M NaCl and 5 mM DTT (Roth) in TE Buffer) and incubated overnight at 65°C. The next day 200 µg of RNAse A (Thermo Fisher Scientific) were added and the samples were incubated at 37°C for 1 h. 100 µg of Proteinase K (Sigma) were subsequently added, followed by a 1 h incubation at 50°C. Phenol/chloroform/isoamyl alcohol (Roth) was added to each sample in a 1:1 ratio, the mixture was vortexed for 1 min and subsequently centrifuged at 16,000 x g for 10 min at room temperature. The aqueous phase was transferred to a new tube, 1 ml 100% EtOH, 90 µl 5 M NaCl and 1µl Pellet Paint (Merck) was added to each sample, mixed, and incubated at -80°C for 1 h. DNA was pelleted by centrifugation for 16,000 x g for 15 min at 4°C, pellets were washed twice with 70% EtOH, air-dried and resuspended in 50 µl water.
Sequencing libraries were prepared from both sorted and unsorted cell populations for each of the three independent screen replicates. Genomic DNA from frozen cell pellets was isolated by Phenol/Chloroform extraction. Briefly, cell pellets were thawed and resuspended in 250 µl of Lysis buffer (1% SDS (Thermo Fisher Scientific), 0.2 M NaCl and 5 mM DTT (Roth) in TE Buffer) and incubated overnight at 65 °C. 200 µg of RNAse A (Thermo Fisher Scientific) were added to the sample and incubated at 37 °C for 1 h. 100 µg of Proteinase K (Sigma) were subsequently added followed by a 1 h incubation at 50 °C. Phenol/Chloroform/Isoamyl alcohol (Roth) was added to each sample in a 1:1 ratio, the mixture was vortexed at room temperature for 1 min and subsequently centrifuged at 16,000 x g for 10 min at room temperature. The aqueous phase was transferred to a new tube, 1 ml 100% EtOH, 90 µl 5 M NaCl and 2 µl Pellet Paint (Merck) was added to each sample, mixed, and incubated at -80 °C for 1 h. DNA was pelleted by centrifugation for 16,000 x g for 15 min at 4 °C, pellets were washed twice with 70% EtOH, air-dried and resuspended in 50 µl H2O. The genomically integrated sgRNA cassette was PCR-amplified to attach sequencing adaptors and sample barcodes. To ensure proper library coverage (300x), a total of 20 µg of each sample were amplified using the ReadyMix Kapa polymerase (Roche) with a total of 25 cycles and an annealing temperature of 56 °C. A relatively low amount of 0.5 µg genomic DNA was amplified per 50 µl PCR reaction since in samples stained with Flow-FISH, PCR amplification was inhibited at higher DNA concentrations. PCR was performed with the primer LR175 in combination with a sample-specific primer which contains a distinct 6-nucleotide barcode to allow sample identification after multiplexed deep sequencing (Primer sequences in Suppl. Table 4, LR178/LR180). Successful amplification was verified on a 1% agarose gel and the reactions were pooled. 1 ml of each pooled PCR was purified using the QIAquick PCR Purification Kit (Qiagen) and the resulting amplicons were loaded on a 1% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen).
CRISPR screen
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Alignment and read counting was performed using MAGeCK (0.5.9.3) with options [count --norm-method control] for all samples together (Li et al. 2014, Li et al. 2015).
Statistical analysis was performed using MAGeCK (0.5.9.3) with options [mle --norm-method control] (Li et al. 2014, Li et al. 2015)
Genome_build: -
Supplementary_files_format_and_content: char_library_dup.txt: Text file containing descriptions of the individual guides used for the screen. As some guides target multiple TSSs, they are duplicated within the file.
Supplementary_files_format_and_content: raw_counts_dup.txt: Raw counts assessed via MAGeCK count. Guides targeting multiple TSSs are duplicated.
Supplementary_files_format_and_content: mageck_mle_dup.txt: Results of the MAGeCK MLE analysis, showing enrichment of individual TSSs within the screen.
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| Submission date |
Jan 19, 2022 |
| Last update date |
Jan 26, 2022 |
| Contact name |
Till Schwämmle |
| E-mail(s) |
Till.schwaemmle@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Otto-Warburg-Laboratory
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| Lab |
Schulz Lab
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| Street address |
Ihnestrasse 63
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| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE194013 |
CRISPR activation screen to identify X-chromosomal Xist activators in male embryonic stem cells |
| GSE194018 |
A role for the GATA transcription factor family in X-chromosome inactivation as direct potent Xist activators |
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| Relations |
| BioSample |
SAMN25124160 |
| SRA |
SRX13833437 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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