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| Status |
Public on Apr 22, 2021 |
| Title |
HA5-Early_IgG_P1_CUT&RUN |
| Sample type |
SRA |
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| Source name |
randomly integrated HOTAIR cDNA deleted for the first 300 bp by lentiviral transduction of HA5-Early cell line (PMID: 23358853)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HA5-Early
variant_id(strain): P
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| Treatment protocol |
CUT&RUN protocol (Skene et al 2018); Antibodies: H3K27ac (Millipore, ref MABE647, lot VP1910310); H3K4me2 (Millipore, ref 07-030, lot DAM1503382); H3K9me2 (Active Motif, ref 39239, lot 34718002); Lsd1 (Diagenode, ref C15410067, lot A57-00234P); IgG (Invitrogen, ref 31235, lot UJ2871342). Detailed protocol: For each cell line two biological replicates were used. Cells were grown up to 90% confluence in 10 cm² petri plate in MEM-alpha medium supplemented with non-essential amino-acids and sodium pyruvate, at 37°C, 5% CO2.
For histone marks cells were scrapped from plates collected by centrifugation. Pellets were resuspended in media with 10% DMSO, slowly frozen and kept at -80°C. For CUT&RUN experiment, cells were quickly thawed in 37°C and collected by centrifugation. For Lsd1, and IgG we proceeded directly with fresh cells scrapped from culture dishes and collected by centrifugation.
Cell pellets were resuspended in Wash buffer and washed twice before resuspending in volume 10^6 cells/100 µl. For each IP sample, 100 µl of cell suspension were aliquoted to 900 µl of Wash Buffer in 1.5ml eppendorf tube containing 10 µl of Concanavalin A beads (Epicypher) previously washed in Bead activation buffer (20mM HEPES pH 7.9, 10mM KCl,1mM CaCl2, 1mM MnCl2). Cells were incubated with beads for 10 min with rotation at 15-20 rpm at room temperature (RT). Beads were separated from supernatant on a magnetic stand and resuspended in 50 µl of the antibody diluted in Antibody buffer (wash buffer with 0,1% digitonin and 2mM EDTA) (1:100 dilution for all antibodies and 1:1100 for IgG control). The beads were incubated with subtle agitation for 10 min for histone marks and 1h for Lsd1, Ezh2 and IgG antibodies at RT. Afterwards the beads were washed twice with Digitonin buffer (Wash buffer with 0,1% digitonin) and resuspended in pAG-MNase (CUTANA Epicypher) diluted 20 times in Digitonin buffer. After 10 min incubation with slight agitation at RT, beads were washed twice and resuspended in Digitonin buffer and chilled to 0°C (ice) for 5 min. To activate MNase, 2 µl of 100 mM CaCl₂ were added and incubated for additional 30 min on ice. To stop the reaction, 100 µl of Stop buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 50ug/ml RNase A, 50ug/ml Glycogen, 0.1% Digitonin, 10 pg/ml Spike-in DNA) was added and beads were incubate for 10 min at 37°C followed by centrifugation at 16k rcf at 4°C for 5 min. The supernatant was recovered and DNA fragments were purified using NEB Monarch PCR & DNA purification kit with protocol enriching for short DNA fragments. DNA quantity was analyzed using Qubit HS DNA kit.
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| Growth protocol |
Cells were grown up to 90% confluence in 100 cm² petri plate in MEM-alpha medium supplemented with non-essential amino-acids and sodium pyruvate, at 37°C, 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq libraries were prepared with TruSeq ChIP Sample Prep Kit (IP-202-1012, Illumina)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: CUT&RUN
mapping with Bowtie2 (bowtie2 -I 10 -X 700 –no-mixed)
filtering reads (samtools view -q 30 --F 0x100 -F 0x800)
peak calling HMCan (smallBinLength : 50, largeBinLength : 25000, pvalueThreshold : 0.01, PosteriorProb : 0.7, CNAnormalization : False, RemoveDuplicates : False, pairedEnds : True, iterationThreshold : 5, finalThreshold : 0, maxIter : 20 )
peak calling EPIC2 (--keep-duplicates –chromsizes [file with chromosome size, from chrI to chrM – no scaffolds])
merging replicates peaks for each condition, for a given histone mark/protein (bedtools merge)
merging peaks of all strain, for a given histone mark/protein (bedtools merge)
Genome_build: GRCh38
Supplementary_files_format_and_content: tab delimited file (GFF format), genomic location of peaks, and the strain(s) in which they were called
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| Submission date |
Dec 09, 2020 |
| Last update date |
Apr 22, 2021 |
| Contact name |
Marina Pinskaya |
| E-mail(s) |
marina.pinskaya@curie.fr
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| Organization name |
Institut Curie
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| Street address |
rue d'Ulm
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| City |
Paris |
| ZIP/Postal code |
75 |
| Country |
France |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE106517 |
HOTAIR lncRNA role in epithelial-mesenchymal transition |
| GSE162937 |
Profiling of Lsd1 and chromatin marks in epithelial cells overexpressing HOTAIR variants |
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| Relations |
| BioSample |
SAMN17040335 |
| SRA |
SRX9661316 |
| 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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