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Enhancer Release and Retargeting” Activates Disease Susceptibility Genes (GRO-Seq)

ABSTRACT: Purpose: We used Global run-on sequencing, a gold standard assay to measure transcriptional activity to examine the change of genome transcription in the absence of RAD21 subunit of cohesin, a critical chromatin architectural protein complex. In particularl, we tested how the loss of chromatin looping regulator cohesin would impact the enhancers and their target promoter transcription. Methods: Transfection of small interfering RNAs (siRNAs) into MCF-7 cells was performed using Lipofectamine 2000 (Life technologies) following manufacturer's instructions. For all experiments, two rounds of siRNA transfection (40nM each time) were performed to achieve higher efficiency. The siRNAs used in this study include: Qiagen Negative Control siRNA (Qiagen Cat# 1027310) and/or Sigma Mission siRNA universal control #2 (SIC002); siRAD21 (Sigma SASI_Hs02_00341219 and SASI_Hs01_00195799). The transfected MCF7 cells were subjected to GRO-seq experiments. GRO-Seq experiments were performed as previously reported (Li et al., 2013, Nature). Briefly, ~10-20 millions of MCF-7 cells were washed 3 times with cold PBS and then sequentially swelled in swelling buffer (10mM Tris-Cl pH7.5, 2mM MgCl2, 3mM CaCl2) for 5 min on ice, harvested, and lysed in lysis buffer (swelling buffer plus 0.5% NP-40 and 10% glycerol). The resultant nuclei were washed one more time with 10mL lysis buffer and finally re-suspended in 100uL of freezing buffer (50mM Tris-Cl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For the run-on assay, re-suspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of SUPERase-IN, 1% sarkosyl, 500uM ATP, GTP, and Br-UTP, 2uM CTP) and incubated for 5 min at 30°C . The resultant nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Life Technologies) following manufacturer’s instructions. NRO-RNA was fragmented to ~300-500nt by alkaline base hydrolysis on ice and followed by treatment with DNase I and Antarctic phosphatase. These fragmented Br-UTP labeled nascent RNA was then immunoprecipitated with an anti-BrdU argarose beads (Sc32323ac, Santa Cruz Biotechnology) in binding buffer (0.5XSSPE, 1mM EDTA, 0.05% tween) for 3 hrs at 4°C with rotation. Subsequently, T4 PNK was used to repair the end of the immunoprecipitated BrU-NRO-RNA, at 37°C for 1hr. The RNA was extracted and precipitated using acidic phenol-chloroform. cDNA synthesis was performed as per a published method (54) with few modifications. The RNA fragments were subjected to poly-A tailing reaction by poly-A polymerase (NEB) for 30 min at 37°C. Subsequently, reverse transcription was performed using oNTI223 primer and superscript III RT kit (Life Technologies). The cDNA products were separated on a 10% polyacrylamide TBE-urea gel and only those migrated between ~100-500bp were excised and recovered by gel extraction. After that, the first-strand cDNA was circularized by CircLigase (Epicentre) and re-linearized by APE1 (NEB). Re-linearized single strand cDNA (sscDNA) was separated by a 10% polyacrylamide TBE gel as described above and the product of needed size was excised (~170-400bp) for gel extraction. Finally, sscDNA template was amplified by PCR (usually between 10-14 PCR cycles) using the Phusion High-Fidelity enzyme (NEB) according to the manufacturer’s instructions. The resulted library was subjected to deep sequencing. Results: Using an optimized data analysis workflow, we generated three biological replicates of GRO-seq in the presence or absence of cohesin, and with or without treatment by estrogen.

ORGANISM(S): Homo sapiens

PROVIDER: GSE115603 | GEO | 2020/08/31

REPOSITORIES: GEO

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

Project description:The discovery of small open reading frames (smORFs) encoding for polypeptides (SEPs; <100aa) highlights that the coding capacity of the genomes has been underestimated. Most ORF-finding algorithms have historically set a minimum threshold length of 100 aa. As consequence, some transcripts encoding for SEPs, were erroneously discarded or classified as non-coding RNAs (ncRNAs). With this experiments we try to experimentally assess the existence and complexity of these small proteins. The experimental design includes: After growing each Mycoplasma for 6h at 37°C, cells were washed twice with PBS and lysed with 700 µl of Qiazol buffer. Then, samples were lysed with 700 µl of Qiazol buffer. RNA extractions were performed by using the miRNeasy mini Kit (Qiagen) following the instructions of the manufacturer. Libraries for RNA-seq were prepared following directional RNA-seq library preparation and sequencing. Briefly, 1 µg of total RNA was fragmented to ~100-150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5’ and 3’ ends of the RNA available for adapter ligation. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer's protocol. In summary, 3’ adapters and subsequently 5’ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3’ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit. Finally, size selection of the libraries was performed using 6% Novex® TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 100 to 130 bp were cut from the gel, and cDNA was precipitated and eluted in 10 µl of elution buffer. Double-stranded templates were cluster amplified and sequenced on an Illumina HiSeq 2000.

Project description:Bacteria was grown at 30C in 3 different conditions, i.e. SYN (syngas and minimal medium- ATCC no 1789), AC (0.3% acetate in minimal medium- ATCC no 1789) and TSB (tryptic soy broth). After harvesting by centrifugation, O. carboxidovorans pellets (1g) were lysed in 100 mM Tris-Cl pH 8.0, 2% Triton X-100, 2.6 mg/ml sodium azide, 8 mM PMSF by sonication on ice (4 pulses of 15 s duration each). For each condition of growth 4 samples (1g pellets) were separately treated (i.e. lysed and processed further). The supernatants were treated with 50% cold TCA, and the precipitated protein washed with acetone. The pellets were resuspended in solubilization buffer (7M urea, 20 mM tris-Cl, pH 8.0, 5 mM EDTA, 5 mM MgCl2, 4% CHAPS), and protein concentration was determined using the Plus One 2-D Quant Kit (Amersham) following the manufacturers instructions. Protein samples from each treatment were stored at -80 C. One hundred micrograms of each protein sample was resuspended in 0.1 M ammonium bicarbonate, 5% HPLC grade ACN, reduced in 5 mM DTT (65 C, 5 min), alkylated in 10 mM iodoacetamide (30 C, 30 min), and then trypsin digested until there was no visible pellet (1:50 w/w 37 C, 16 h). Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA) and eluted using a 0.1% TFA, 95% ACN solution. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ?l of 0.1% formic acid. Peptides were separated by strong cation exchange (SCX) liquid chromatography (LC) followed by reverse phase (RP) LC coupled directly in line with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). 2DLC ESI MS/MS was done exactly as described (1). All searches were done using TurboSEQUEST (Bioworks Browser 3.2; Thermo Electron). Mass spectra and tandem mass spectra were searched against all annotated proteins from the strain OM5 including all the annotated plasmid-encoded proteins. Cysteine carbamidomethylation and methionine oxidation (single and double) were included in the search strategy. We used the reverse database functionality in Bioworks 3.2 and searched MS2 data against a reversed OM5 database using identical search criteria.

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Enhancer Release and Retargeting” Activates Disease Susceptibility Genes (GRO-Seq)

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