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| Status |
Public on Jun 23, 2021 |
| Title |
P-TEFb rep2 |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
strain: E14
ip antibody: CDK9 sc-484
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| Growth protocol |
Cells were cultured on 0.1% gelatin-coated surfaces with KO-DMEM medium, 10% FCS, 5% knockout serum replacement, nonessential amino acids, L-glutamine, 2-mercaptoethanol, antibiotics (Invitrogen), and 1000 U/ml of leukaemia inhibitory factor (Abcam).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.2 J/cm2 of UV light in a Strataliker 2400 (Stratagene) with bulbs emitting at 254nm, and 6-10x107 cells per IP were lysed in 1mL of Lysis buffer. Lysates were syringed with a 27G needle and sonicated for 3x 10s pulses with a Diagenode Picoruptor. Lysates were incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/mL of DNase Turbo and of 20 U/mL of RNase I. IPs were set up using 5ug of antibody as previously described in the iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and 1M Urea), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50mM MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m mM MgCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in NuPAGE loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellμlose membrane (Hybond, GE Healthcare) washed 2x with 1x PBS and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol:chloroform extraction and ethanol precipitation overnight at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μll Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Reverse transcription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation for 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated by running samples on a precast 6% TBE-urea gel at 180 V 40 min and isolating bands at 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol:chloroform extraction and ethanol precipitation overnight at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl; Circligase II [Epicentre]) transferred into a 0.2ml tube and incubated for 1 h at 60°C for circularization. Samples were incubated in 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), the oligo was annealed in a decreasing temperature gradient and circular cDNA re-linearized by incubating samples with 2 μl BamHI (Fast Fermentas). cDNA was isolated by phenol:chloroform extraction and ethanol precipitation overnight at -20°C and the pellet resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa (10 μM each); 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using a KAPA kit according to the manufacturer's instructions.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
protein crosslinked RNA
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| Data processing |
Fastq files were demultiplexed and aligned using the iCount server. The unique molecular identifiers (UMIs) were registered and experimental barcodes removed before mapping the sequences to mm9 using Bowtie version 0.12.7 (command line: -v 2 -m 1 -a --best --strata.
Reads indicative of PCR duplicates (reads mapping to the same position with the same UMI) and reads aligning to multiple positions in the genome were removed.
Reads were filtered to remove any crosslinks within 50 nt of a repeatMasker-annotated repeat or a short non-coding RNA.
Genome_build: mm9
Supplementary_files_format_and_content: bed files of crosslinks mapping to unqiue positions in the genome filtered for crosslinks mapping to repeat regions or small ncRNAs.
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| Submission date |
May 15, 2020 |
| Last update date |
Jun 23, 2021 |
| Contact name |
Richard Jenner |
| E-mail(s) |
r.jenner@ucl.ac.uk
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| Organization name |
UCL Cancer Institute
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| Department |
Cancer Biology
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| Lab |
Regulatory Genomics
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| Street address |
72 Huntley Street
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| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE150677 |
Nascent RNA antagonises the interaction of a set of regulatory proteins with chromatin |
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| Relations |
| BioSample |
SAMN14933917 |
| SRA |
SRX8349989 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4556268_PTEFb_PCR2.bed.gz |
759.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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