2024-03-15
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA13409754
External Id
KING'S COLLEGE LONDON
INSDC center name
2022-03-15T17:15:35Z
INSDC last update
public
INSDC status
ERS11012223
SRA accession
E-MTAB-11544:ChIPseq_input_no_IAA_rep1
Submitter Id
ArrayExpress
broker name
Ptbp1-mAID
cell_line
mouse embryonic stem cell
cell_type
house mouse
common name
Protocols: Ptbp1-mAID mouse embryonic stem cells (Ptbp1-mAID ESCs) with a doxycycline (Dox) inducible OsTIR1 transgene and C-terminal mAID tags added to both Ptbp1 alleles were cultured in 15-cm gelatinized tissue culture dishes in 2i medium containing a 1:1 mixture of Neurobasal (Thermo Fisher Scientific, cat#21103049) and DMEM/F12 (Sigma, cat#D6421) media supplemented with 100 units/ml PenStrep (Thermo Fisher Scientific, cat#15140122), 1 μM PD03259010 (Cambridge Bioscience, cat#SM26-2), 3 μM CHIR99021 (Cambridge Bioscience, cat#SM13-1), 0.5 mM L-glutamine (Thermo Fisher Scientific, cat#25030024), 0.1 mM β-mercaptoethanol (Sigma, cat#M3148), 1,000 units/ml ESGRO LIF (Millipore, cat#ESG1107), 0.5× B-27 supplement without vitamin A (Thermo Fisher Scientific, cat#12587010) and 0.5× N2 supplement. N2 100× stock was prepared using DMEM/F12 medium as a base and contained 5 mg/ml BSA (Thermo Fisher Scientific, cat#15260037), 2 µg/ml progesterone (Sigma, cat#P8783), 1.6 mg/ml putrescine (Sigma, cat#P5780), 3 µM sodium selenite solution (Sigma, cat#S5261), 10 mg/ml apo-transferrin (Sigma, cat#T1147), and 1 mg/ml insulin (Sigma, cat#I0516-5ML) and stored in single-use aliquots at -80°C. After seeding ~1×10^7 cells per dish and a 24-hour incubation at 37°C, 5% CO2, OsTIR1 expression was induced by treating the cultures with 2 μg/ml Dox for another 24 hours. The \"plus IAA\" samples were then incubated with 0.5 mM IAA (indole-3-acetic acid) for 4 or 24 hours, and the \"no IAA\" samples, without IAA for the same period of time. To extract total RNA, we added 1 ml TRIzol reagent (Thermo Fisher Scientific, cat#15596018) per 0.5-1×10^7 cells, mixed well and incubated at room temperature for 2-3 minutes. The organic and aqueous phases were separated by the addition of 0.2 ml of chloroform and centrifuging the samples at room temperature for 10 min at 18,000×g. The upper, aqueous phase was transferred to a fresh tube containing 0.5 ml of a 1:1 mixture of acidic phenol and chloroform, mixed well, and then centrifuged as above. The aqueous phase (~0.5 ml) was transferred to a fresh tube and precipitated with 0.5 ml of isopropanol. RNA pellets were washed with 1 ml of 70% ethanol, dried and rehydrated in 90 µl of nuclease-free water. Traces of genomic DNA were removed by incubating the samples with 10 µl of 10× RQ1 DNase buffer (Promega), 4 µl of RQ1 DNase (Promega) and 1 µl of Recombinant RNasin (Promega) at 37°C for 30 min, followed by acid phenol-chloroform extraction and ethanol precipitation. Chromatin-associated RNA fraction was prepared as follows. Cells were washed twice with 20 ml of ice-cold PBS per dish, scraped off, and collected by centrifugation at 420×g for 5 min at 4°C. The pellet was resuspended in 4 ml of ice-cold HLB+N buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2.5 mM MgCl2 and 0.5% NP-40) and incubated for 5 min on ice. The samples were then underlaid with 1 ml of ice-cold HLB+NS (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40 and 10% sucrose) and spun down at 420×g for 5 min. Nuclear pellets were resuspended in 125 µl of ice-cold NUN1 buffer (20 mM TrisHCl pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol), transferred to fresh 1.5-ml microcentrifuge tubes, mixed with 1.2 ml of ice-cold NUN2 buffer (20 mM HEPES-KOH pH 7.6, 0.2 mM EDTA, 300 mM NaCl, 7.5 mM MgCl2, 1% NP-40, 1 M urea), and vortexed at a maximum speed. The samples were incubated on ice for 15 min, vortexing the tubes briefly every 3-4 minutes, and centrifuged at 16,000×g for 10 min at 4°C. Chromatin pellets were resuspended in 200 µl of HSB (10 mM, Tris-HCl, pH 7.5, 500 mM NaCl and 10 mM MgCl2) and incubated with 25 µl of TURBO DNase at 37°C for 10 min and then with 20 µl of Proteinase K (Thermo Fisher Scientific, cat#EO0491) for another 10 min and extracted with TRIzol reagent as recommended by the manufacturer. The TURBO DNase, Proteinase K, and TRIzol steps were repeated in total three times. Samples for mNET-seq analysis were prepared as in PMID:26844429. Briefly, chromatin pellets prepared as described above and digested with 40 gel units/mL of micrococcal nuclease (MNase; New England Biolabs, cat#M0247S) in MNase reaction buffer at 37°C for 3 min. Samples were immunoprecipitated for 1 hour with the CMA601 antibody recognizing the CTD-phosphorylated and unphosphorylated forms of Pol II (MBL Life science cat#MABI0601; 20 µg per dish) and 400 µl Dynabeads M280 sheep Anti-mouse IgG (Thermo Fisher Scientific, cat#11202D). Bead-bound Pol II complexes were washed with NET-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NP-40) 6 times. After IP, the beads were treated with 1 unit/µl T4 Polynucleotide Kinase (T4 PNK; New England Biolabs, cat#M0236S) in 1× T4 PNK buffer with 1 mM ATP at 37°C for 6 min and then washed with NET-2 buffer once more. TRIzol was directly added to the beads to purify RNAs according to the manual. RNAs were separated by electrophoresis in a 6% Novex™ TBE-Urea Gel (Thermo Fisher Scientific cat#EC68652BOX) for 5 min at 280 V constant. RNAs migrating between the xylene cyanol and bromophenol blue dyes were excised from the gel. Gel slices were fragmented by transferring them to 0.5 ml tubes with 25G needle-pierced holes in the bottom, inserted into 1.5-ml collection tubes, and centrifuging at 16,000×g for 1 min at 4°C. The 0.5 ml tubes were then discarded and 400 µl of gel elution buffer (1M NaOAc, 1 mM EDTA, pH 8.0) was added to the 1.5-ml tubes. The tubes were shaken at 1,200 rpm, 25°C for 1 hour. Gel fragments were removed by passing the suspensions through Spin-X columns (Costar, cat#8160). RNAs in the flowthrough fractions (~400 µl) were precipitated by adding 0.5 µl GlycoBlue (Thermo Fisher Scientific, cat#AM9515) and 1 ml 100% ethanol. The RNA pellets were washed with 70% EtOH, air-dried and rehydrated in 8 µl nuclease-free water. Chromatin immunoprecipitation (ChIP) protocol was modified from PMID:20028857. Briefly, we fixed 15-cm dish cultures by adding formaldehyde directly to the 2i medium at a final concentration of 1% and incubating for 10 min at room temperature. The samples were then quenched by 125 mM glycine (final concentration), washed with ice-cold PBS, and scraped off to isolate nuclei in a 5 mM PIPES pH 8.0, 85 mM KCl and 0.5% NP40. Nuclei were resuspended and lysed in 3 ml of 1% SDS, 10 mM EDTA and 50 mM Tris/HCl pH 8.1. Lysates were then sonicated to yield 150–300 bp DNA fragments using a Bioruptor Plus system equipped with a refrigerated sonication bath (Diagenode; 30 seconds \"ON\" and 30 seconds \"OFF\" cycles, delivered at HIGH setting). A total of 100 μg of DNA per sample (estimated by a NanoDrop spectrophotometer) was then immunoprecipitated using 5 μg of either monoclonal rabbit anti-PTBP1 (Abcam, cat#ab133734) or rabbit IgG control. The complexes were captured on Dynabeads Protein G for Immunoprecipitation (Thermo Fisher Scientific, cat#10003D). Following immunoprecipitation, beads were washed 3 times with NET-2 buffer and the chromatin was eluted by incubating the beads in 1% SDS, 10 mM Tris, pH 7.5, 1 mM EDTA and 250 mM NaCl at 65°C for 20 min. The eluates were decrosslinked at 65°C overnight and ethanol-precipitated. The recovered material was treated with 30 μg of Proteinase K (Roche Diagnostics, Mannheim, Germany) in 25 μl of Proteinase K buffer (1.25% SDS, 50 mM Tris, pH 7.5, 25 mM EDTA, pH 8), diluted with 100 μl of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA), extracted with phenol/chloroform/isoamyl alcohol (25:24:1), and precipitated with ethanol. The pellets were dissolved in nuclease-free water. Strand-specific poly(A)+ RNA-seq libraries were prepared from total RNA samples using a NEBNext Ultra II mRNA Kit (New England Biolabs, cat#E7765S), as recommended. Libraries were amplified using unique dual indexing and normalized using a Qubit (Thermo FIsher Scientific). Library size profiles were analyzed using a 2200 or a 4200 TapeStation (Agilent). Libraries were pooled, diluted to 10 nM, denatured and further diluted prior to loading on the sequencer. To prepare strand-specific ribodepleted RNA-seq libraries for chromatin-associated RNAs, samples were rRNA-depleted using a NEBNext rRNA Depletion Kit (New England Biolabs, cat#E6350S). rRNA-depleted samples were used for RNA library preparation using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer’s protocol. Libraries for mNET-Seq were prepared from gel-purified RNA fragments using a NEBNext Small RNA Library Prep Kit (New England Biolabs, cat#E7300S). In both cases, pooled libraries were diluted, denatured and sequenced using a HiSeq4000 paired-end format. ChIP eluates and chromatin input samples were quantified using a Qubit and analyzed on a TapeStation (Agilent, dsDNA HS Assay). Automated library preparation was performed using the Apollo prep system (Wafergen, PrepX ILMN 32i, 96 sample kit) and standard Illumina multiplexing adapters following manufacturer’s protocol up to pre-PCR amplification. Libraries were PCR amplified (18 cycles) using the NEBNext High-Fidelity 2× PCR Master Mix (NEB) and single indexing primers. Individual libraries were normalized using Qubit and analyzed on a TapeStation, pooled, diluted, denatured and sequenced using a NovaSeq6000 paired-end format.
description
embryo
developmental stage
normal
disease
dox-OsTIR1; Ptbp1-mAID
genotype
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
inner cell mass
organism part
Mus musculus
scientific_name
ChIPseq_input_no_IAA_rep1
title