title: Human chronic lymphocytic leukemia patient DR0312


title: Human chronic lymphocytic leukemia patient DD0280

status: Public on Mar 24 2014

type: RNA

city: MILAN

first: Luca

last: Agnelli

address: Venezian 1

department: Department of Advanced Diagnostics

country: Italy

institute: IRCCS Istituto Nazionale dei Tumori

description: Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer (Becton Dickinson & Co, Sunnyvale, CA) with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome (Becton Dickinson). If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody (mAb) treatment (Becton Dickinson) followed by magnetic beads (Goat Anti-Mouse IgG Dynabeads, Dynal Biotech ASA, Oslo, Norway).

hyb_protocol: The fragmented labeled single-stranded DNA target was hybridized for 16 hours and 30 minutes at 45°C on GeneChip® Gene 1.0 ST array according to the standard Affymetrix protocol.

scan_protocol: Washing and scanning were performed using GeneChip System of Affymetrix (GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450 and GeneChip Scanner 7G).

data_processing: The raw intensity expression values were processed by Robust Multi-array Average (RMA) complete procedure with the re-mapped Chip Definition Files (CDF) from BrainArray libraries version 15.0.0 available at http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/15.0.0/entrezg.asp.

label: biotin

molecule: total RNA

organism: Homo sapiens

label_protocol: 5.5 micrograms of single-stranded DNA target obtained from 100 ng of purified total RNA was fragmented and then labeled using the WT Terminal Labeling Kit according to the standard Affymetrix protocol (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual).

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions (Gibco BRL).

tag: vh status = igvh mutational status (fais et al. j clin invest, 1998)

value: mut


title: Human chronic lymphocytic leukemia patient CR0203

treatment_protocol: Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer (Becton Dickinson & Co, Sunnyvale, CA) with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome (Becton Dickinson). If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody (mAb) treatment (Becton Dickinson) followed by magnetic beads (Goat Anti-Mouse IgG Dynabeads, Dynal Biotech ASA, Oslo, Norway).

value: unmut


title: Human chronic lymphocytic leukemia patient DE0279


title: Human chronic lymphocytic leukemia patient FD0404


title: Human chronic lymphocytic leukemia patient CP0104


title: Human chronic lymphocytic leukemia patient CS0030


title: Human chronic lymphocytic leukemia patient DN0063


title: Human chronic lymphocytic leukemia patient DF0319


title: Human chronic lymphocytic leukemia patient CR0297


title: Human chronic lymphocytic leukemia patient CM0246


title: Human chronic lymphocytic leukemia patient CS0290


title: Human chronic lymphocytic leukemia patient CR0159


title: Human chronic lymphocytic leukemia patient DA0029


title: Human chronic lymphocytic leukemia patient DG0186


title: Human chronic lymphocytic leukemia patient CM0289


title: Human chronic lymphocytic leukemia patient CM0057


title: Human chronic lymphocytic leukemia patient CA0345


title: Human chronic lymphocytic leukemia patient CI0005


title: Human chronic lymphocytic leukemia patient CE0399


title: Human chronic lymphocytic leukemia patient CC0244


title: Human chronic lymphocytic leukemia patient BR0218


title: Human chronic lymphocytic leukemia patient CC0311


title: Human chronic lymphocytic leukemia patient CD0310


title: Human chronic lymphocytic leukemia patient CG0166


title: Human chronic lymphocytic leukemia patient BV0229


title: Human chronic lymphocytic leukemia patient CA0101


title: Human chronic lymphocytic leukemia patient BS0214


title: Human chronic lymphocytic leukemia patient CA0160


title: Human chronic lymphocytic leukemia patient BC0047


title: Human chronic lymphocytic leukemia patient BF0316


title: Human chronic lymphocytic leukemia patient BB0245


title: Human chronic lymphocytic leukemia patient CG0204


title: Human chronic lymphocytic leukemia patient BP0326


title: Human chronic lymphocytic leukemia patient BG0236


title: Human chronic lymphocytic leukemia patient CA0058


title: Human chronic lymphocytic leukemia patient CE0078


title: Human chronic lymphocytic leukemia patient BF0217


title: Human chronic lymphocytic leukemia patient CP0109


title: Human chronic lymphocytic leukemia patient BE0192


title: Human chronic lymphocytic leukemia patient BV0176


title: yFR116_newAbTFIIBvsInput_rep1

type: genomic

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

country: Canada

institute: IRCM

description: Biological replicate 1 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells.

hyb_protocol: Hybridization: Resuspend colored pellet in 110uL hybridization buffer (100uL DIGEasy Buffer, 5uL 10mg/mL Salmon Sperm DNA, 5uL 8mg/mL yeast tRNA). Put at 95?C for 5 minutes. Put at 42?C for 10 min (Keep samples at 42?C while waiting for hybridization). Follow standard Agilent chamber hybridization protocols. Incubate overnight (16-24h) at 42?C in Agilent hybridization oven at 20 rpm rotation speed.  Post-Hybridization Washes: Separate coverslip and slide in Wash buffer #1. Wash slides in Wash buffer #1 for 5 minutes at room temperature on an orbital shaker (cover the dish to avoid light on the slides). Wash in 31C Wash buffer #2 for 5 minutes on an orbital shaker. Take slides out of the buffer slowly to dry them. Wash Buffer #1: 6x SSPE, 0.005% N-lauroylsarcosine. Wash Buffer #2: 0.06x SSPE.  Stripping: Stripping is done in 1L of 5 mM Potassium phosphate buffer pH 6.6 after scanning of the slides. 1. Use a crystallization dish, and a metallic slide rack (use weighing spatulas to lift the rack from the bottom of the dish and avoid direct contact with the hot surface). Pour the stripping buffer, submerge the rack with slides (from 1 to 10) and heat it up slowly on heating plate until the liquid boils with big bubbles. It usually takes 15 to 20 minutes. Do not overboil it. 2. Remove the rack and briefly rinse in a container with water. Slowly remove the rack from the liquid. Cy5 channel will be stripped much better than Cy3, but this does not affect the performance of subsequent hybridizations.  Stock solution of 1M Potassium phosphate buffer, pH 6.6: Mix 3.81 ml of 1M K2HPO4 + 6.19 ml of 1M KH2PO4. Dilute 5 ml of this 1M solution in 1L Milli-Q water to obtain 5mM Potassium phosphate buffer, pH 6.6.

scan_protocol: Standard_Innopsys_Yeast4x180K  Set the scan as follow:  pixel size = 2Âµm;  speed = 20;  Scan Mode = Normal;  Acquisition Mode = Simultaneous;  Focus = Auto.    Adjust manually the detection gain for 635 and 532 wavelengths in order to have less than 5% spot saturation.

data_processing: Correction = Foreground-Background  Normalisation = limma loess (subgrid)

molecule: genomic DNA

organism: Saccharomyces cerevisiae

label_protocol: Blunting: Do everything on ice! Add 70uL of the blunting solution (11uL NEB Buffer #2, 0.5uL BSA 10mg/mL, 0.5uL 20mM dNTPs, 0.2uL T4 DNA Polymerase 3U/uL, 57.8uL ddH2O) to 40uL of the resuspended DNA obtained at the end of the cleanup step. For WCEs, use 2uL + 38uL ddH2O instead of 40uL. Incubate 20 minutes at 12C.    Add 11.5uL 3M NaOAc, pH 5.2, and 0.5uL Glycogen 20mg/mL and vortex briefly.    Extract with 120uL Phenol/Chloroform/Isoamyl alcohol (25:24:1). Transfer 110uL to a new tube, and add 230uL -20C 100% EtOH. Put at -80C for 30min, then spin 20 minutes at 4C. Wash the pellet with 500uL -20C 70% EtOH and spin 15 minutes at 4C. Resuspend the pellet in 25uL ddH2O and place on ice.    Ligation: Thaw everything on ice, and do all manipulations on ice. Add 25uL of ligase mix (10uL 5x T4 Ligase Buffer, 6.7uL 15uM unidirectional linkers, 0.5uL T4 DNA Ligase, High Concentration, 8uL ddH2O) to the resuspended pellet from the blunting step. Incubate overnight at 16C.    Add 6uL 3M NaOAc, pH 5.2, and vortex. Add 130uL -20C 100% EtOH and centrifuge 20 minutes at 4C. Wash the pellet (should be a smear on the side of the tube) with 500uL -20C 70% EtOH. Spin 15 minutes at 4C. Dry the pellet and resuspend in 25uL ddH2O.    Labeling: Add 15uL of PCR Labeling mix (4uL 10x ThermoPol Buffer, 2uL 5mM aa-dUTP mix, 1.25uL Oligo FR1, 7.75uL ddH2O) to the pellet from the ligation step. Start the PCR program, and during the first step (4 minutes at 55C), add 10uL of the polymerase mix (1uL 10x ThermoPol buffer, 1uL Taq Polymerase 5U/uL, 0.01uL Pfu Polymerase Turbo 2.5U/uL, 8uL ddH2O).  PCR cycling conditions:  1) 5:00 at 55C  2) 5:00 at 72C  3) 2:00 at 95C  4) 0:30 at 95C  5) 0:30 at 55C  6) 1:00 at 72C  7) Repeat steps 4-6 31 more times.  8) 4:00 at 72C  9) Forever at 4C    Purify PCR reactions with Qiagen PCR Cleanup kits, following their protocol BUT use a phosphate wash buffer and a phosphate elution buffer instead of their buffers PE and EB, respectively. Also, wash the columns only once, and elute twice with 30uL elution buffer. Speed-vac the eluate to dryness.    Resuspend the pellets with fresh 4.5uL 0.1M Sodium Carbonate Buffer, pH 9.0. Add 4.5uL of the appropriate NHS-ester Cy-dye. Fresh tubes of NHS-ester Cy-dye should be resuspended in 73uL DMSO. Mix well, and incubate at room temperature in the dark for one hour.    Add 35uL 0.1M NaOAc, pH 5.2. Purify using the Qiagen PCR Cleanup protocol using all their buffers, but wash only once and elute twice in 30uL. It is a good idea to elute both samples of a pair (Cy5 and Cy3) in the same tube to avoid DNA loss upon later resuspension. Speed-vac until 10-20uL are left, or to dryness.      Phosphate Wash Buffer (100mL):  0.5mL 1M KPO4, pH 8.5  15.25mL ddH2O  84.25mL 95% EtOH  Solution will be cloudy upon mixing.    Phosphate Elution Buffer (10mL):  0.04mL 1M KPO4, pH 8.5  9.96mL ddH2O    1M KPO4, pH 8.5 (10mL):  9.5mL 1M K2HPO4  0.5mL 1M KH2PO4    1M Sodium Carbonate Buffer, pH 9.0 (100mL):  10.8g Na2CO3  80mL ddH2O  pH to 9.0 with concentrated HCl  Adjust volume to 100mL with ddH2O  DILUTE 1:10 WHEN USING!  Solution will change composition over time. Use only if less than a month old.

growth_protocol: Standard_YEPD_50ml. 50mL YEP+2% Glucose is inoculated at OD600=0.1 from an overnight preculture in the appropriate media and allowed to grow until it reach OD600 0.6-1.0. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde (optional). Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

extract_protocol: Cells are thawed on ice, and resuspended in 700uL lysis buffer with protease inhibitors (solution composition detailed at the end). 500uL of acid-washed glass beads (Sigma) are added, and the cells are lysed using the bead-beater (4x5min cycles, with 5min breaks on ice). The tubes are pierced with a 18-gauge syringe needle to allow transfer of lysate, but not beads. The pierced tubes are put in 2mL collection tubes and spun briefly. The lysate is then homogenized by pipetting and sonicated 4x 20sec at output 7 Watts, with a 1 minute break between sonication cycles. The sonicated lysates is centrifuge for 5 min at 4'C max speed and supernatant used immediately for ChIP (or saved and frozen at -80'C). Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL Pepstatin.

treatment_protocol: Anti-yTFIIB (from Richard Young) antibody. Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 2mL PBS + 0.5% BSA. Anti-yTFIIB (from Richard Young) antibody is added (2uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed using the Dynal magnet system twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

tag: strain

tag: antibody

value: TFIIB


title: yFR1662vsyFR682-bur2D_Gal11-18MYCvsUntagged_rep2

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged bur2D mutant strain (yFR1662) versus the ChIP in untagged bur2D mutant strain (yFR682).

treatment_protocol: 9E10 anti-Myc antibody (home-prepared). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65'C to reverse the crosslinks. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.

value: 9E10 (lab stock by AF)


title: Human chronic lymphocytic leukemia patient BG0163


title: yFR1660vsyFR551-ctk1D_Gal11-18MYCvsUntagged_rep2

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged ctk1D mutant strain (yFR1660) versus the ChIP in untagged ctk1D mutant strain (yFR551).


title: yFR1662vsyFR682-bur2D_Gal11-18MYCvsUntagged_rep1


title: Human chronic lymphocytic leukemia patient AR0242


title: Human chronic lymphocytic leukemia patient AS0012


title: Human chronic lymphocytic leukemia patient AS0407


title: Human chronic lymphocytic leukemia patient AR0131


title: Human chronic lymphocytic leukemia patient AM0067


title: yFR1658vsyFR117-WT_Gal11-18MYCvsUntagged_rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged WT strain (yFR1658) versus the ChIP in untagged WT strain (yFR117).


title: Human chronic lymphocytic leukemia patient AM0220


title: yFR401_9Myc-Srb4vsInput_rep1

description: Biological replicate 1 of 1. Mediator Srb4-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR401 (Srb4-9MYC tagged) cells.


title: Human chronic lymphocytic leukemia patient AM0106


title: Human chronic lymphocytic leukemia patient AG0403


title: yFR1640vsyFR999-srb10D_Gal11-9MYCvsUntagged_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged srb10D mutant strain (yFR1640) versus the ChIP in untagged srb10D mutant strain (yFR999).


title: yFR1658vsyFR117-WT_Gal11-18MYCvsUntagged_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged WT strain (yFR1658) versus the ChIP in untagged WT strain (yFR117).


title: yFR1660vsyFR551-ctk1D_Gal11-18MYCvsUntagged_rep1


title: Human chronic lymphocytic leukemia patient AM0034


title: Human chronic lymphocytic leukemia patient AM0270


title: yFR169_18Myc-MED16vsInput_rep1

description: Biological replicate 1 of 1. Mediator Med16-18MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR169 (MED16-18MYC tagged) cells.


title: yFR116_newAbTFIIBvsInput_rep2

description: Biological replicate 2 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells.


title: Human chronic lymphocytic leukemia patient AG0318


title: Human chronic lymphocytic leukemia patient AG0136


title: Human chronic lymphocytic leukemia patient AG0268


title: Human chronic lymphocytic leukemia patient AC0120


title: yFR1640vsyFR999-srb10D_Gal11-9MYCvsUntagged_rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged srb10D mutant strain (yFR1640) versus the ChIP in untagged srb10D mutant strain (yFR999).


title: yFR178_9Myc-SRB10vsInput_rep1

description: Biological replicate 1 of 1. Mediator Srb10-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR178 (SRB10-9MYC tagged) cells.


title: yFR167_9Myc-SRB11vsInput_rep1

description: Biological replicate 1 of 1. Mediator Srb11-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR167 (Srb11-9MYC tagged) cells.


title: yFR399_9Myc-MED15vsInput_rep1

description: Biological replicate 1 of 1. Mediator Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR399 (MED15-9MYC tagged) cells.


title: yFR116_WT_100WCE_MockvsInput_rep2

description: Biological replicate 2 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells.

treatment_protocol: Rabbit polyclonal antibody from Enzo Life Sciences. Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Antibody is   added (5uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL of cell lysate are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.

value: rabbit IgG


title: yFR119_18Myc-MED19vsInput_rep1

description: Biological replicate 1 of 1. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR119 (Gal11-Med15-9MYC tagged) cells.


title: yFR121_9Myc-MED8vsInput_rep1

description: Biological replicate 1 of 1. Mediator Med8-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR121 (Med8-9MYC tagged) cells.


title: yFR150_18Myc-MED6vsInput_rep1

description: Biological replicate 1 of 1. Mediator Med6-18MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP versus Input DNA in yFR150 (Med6-18MYC tagged) cells.


title: Human chronic lymphocytic leukemia patient AB0350

description: Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer (Becton Dickinson & Co, Sunnyvale, CA) with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome (Becton Dickinson). If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody (mAb) treatment (Becton Dickinson) followed by magnetic beads (Goat Anti-Mouse IgG Dynabeads, Dynal Biotech ASA, Oslo, Norway).


title: yFR117_WT_anti-Srb8vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb8 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb8 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb8 (R. Young)


title: yFR116_WT_100WCE_MockvsInput_rep1

description: Biological replicate 1 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells.


title: yFR117_WT_anti-Srb9vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb9 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb9 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb9 (R. Young)


title: Mesenchymal_A_rep2

city: Prague 2

first: Vojtěch

last: Kulvait

address: U Nemocnice 5

department: Institute of Pathological Physiology

country: Czech Republic

institute: Charles University in Prague - 1st Faculty of Medicine

description:
Sample name: A2
SDF file: 6898481097.sdf
Metrics file: 6898481097Metrics.txt
Annotation file: annotation_6898481097.csv

hyb_protocol: Standard Illumina protocol

scan_protocol: Standard Illumina protocol

data_processing:
Image data was read by beadarray package. Exact processing is described in wiki page https://bitbucket.org/kulvait/blima/wiki/blimatesting.rda. Averaged foreground values GrnF after background correction was used for non-normalized_data.txt (i.e., the Matrix of non-normalized data).
For normalized data was used produced by blima processing pipeline including background correction, variance stabilizing transformation and bead level quantile normalization then probe level summarized.

source_name: mesenchymal cells

label_protocol: Standard Illumina protocol

extract_protocol: RNA extraction using Trizol, as per manufacturer's protocol

tag: medium

value: alfa-MEM

tag: additive

value: FTS

value: FALSE

tag: pdgf-bb

tag: egf

tag: m-csf

tag: insulin

tag: arrayname

tag: group

value: A


title: Mesenchymal_A_rep3

description:
Sample name: A3
SDF file: 6898481097.sdf
Metrics file: 6898481097Metrics.txt
Annotation file: annotation_6898481097.csv


title: yFR399vsyFR116_Gal11-9MYCvsUntagged_noRapa_Rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged strain (yFR399) versus the ChIP in untagged strain (yFR116).

growth_protocol: Standard_YEPD_50ml. 50mL YEP+2% Glucose is inoculated at OD600=0.1 from an overnight preculture in the appropriate media and allowed to grow until it reach OD600=0.6-1.0. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde (optional). Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.


title: yFR117_WT_anti-Srb5vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb5 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb5 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb5 (R. Young)


title: Mesenchymal_E_rep2

description:
Sample name: E2
SDF file: 6898481102.sdf
Metrics file: 6898481102Metrics.txt
Annotation file: annotation_6898481102.csv

value: CellGro

value: HS

value: TRUE

value: E


title: Mesenchymal_A_rep4

description:
Sample name: A4
SDF file: 6898481097.sdf
Metrics file: 6898481097Metrics.txt
Annotation file: annotation_6898481097.csv


title: yFR117_WT_anti-Srb2vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb2 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb2 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb2 (R. Young)


title: Mesenchymal_E_rep4

description:
Sample name: E4
SDF file: 6898481102.sdf
Metrics file: 6898481102Metrics.txt
Annotation file: annotation_6898481102.csv


title: yFR117_WT_anti-Srb7vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb7 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb7 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb7 (R. Young)


title: Mesenchymal_E_rep1

description:
Sample name: E1
SDF file: 6898481102.sdf
Metrics file: 6898481102Metrics.txt
Annotation file: annotation_6898481102.csv


title: Mesenchymal_E_rep3

description:
Sample name: E3
SDF file: 6898481102.sdf
Metrics file: 6898481102Metrics.txt
Annotation file: annotation_6898481102.csv


title: yFR399vsyFR116_Gal11-9MYCvsUntagged_30minRapa_Rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged strain (yFR399) versus the ChIP in untagged strain (yFR116), after 30 min with 200ng/ml rapamycin.

growth_protocol: YEP-Glu_Rapamycin-30min-200ng/ml. YEP+2%Glucose is inoculated at OD600=0.1 from an overnight preculture in the appropriate media and allowed to grow until it reachs OD600=0.6-0.7. Cell cultures are treated with 200ng/ml of Rapamycin and allowed to grow for additional 30 min. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

treatment_protocol:
Rapamycin-30min-200ng/ml
9E10 anti-Myc antibody (home-prepared). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65'C to reverse the crosslinks. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.


title: yFR117_WT_anti-Srb6vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb6 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb6 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb6 (R. Young)


title: yFR117_WT_anti-Srb11vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb11 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb11 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb11 (R. Young)


title: yFR117_WT_anti-Srb4vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb4 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb4 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb4 (R. Young)


title: yFR399vsyFR116_Gal11-9MYCvsUntagged_noRapa_Rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged strain (yFR399) versus the ChIP in untagged strain (yFR116).

value: 9E10 (lab stock by AF)


title: yFR117_Srb4-5uLvsInput

treatment_protocol: ChIP_Srb4 (ab63812).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb4 (Abcam ab63812) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.


title: yFR117_WT_anti-Srb10vsInput_rep1

description: Biological replicate 1 of 1. Mediator occupancy, using anti-Srb10 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: ChIP_Srb10 (R.Young).Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. Anti-Srb10 (from R. Young) antibody is added (5uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors),twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Srb10 (R. Young)


title: yFR117_WT_Rpb3vsInput_Rep1_20130731

description: Biological replicate 1 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

treatment_protocol: Rpb3 W0012 antibody is from neoclone). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009.

value: Rpb3 (W0012 neoclone)


title: yFR117_WT_Rpb3vsInput_Rep2_20130731

description: Biological replicate 2 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.


title: Mesenchymal_D_rep4

description:
Sample name: D4
SDF file: 6898481102.sdf
Metrics file: 6898481102Metrics.txt
Annotation file: annotation_6898481102.csv

value: D


title: 24h-0.5ng-LPS-2_C57BL6

city: Midlothian

first: Sobia

last: Raza

address: Easter Bush Campus

department: The University of Edinburgh

country: United Kingdom

institute: The Roslin Institute

description: 24h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 2

hyb_protocol: Samples were hybridized using Affymetrix hybridization kit materials, according to manufacturers reccomendations

scan_protocol: Hybridisation, washing and scanning of the arrays was performed using the Affymetrix GeneTitan instrument according to the manufacturer’s recommendations.

data_processing:
Data were processed using the Affymetrix Expression Console software version 1.1
probe group file: MoGene-1_1-st-v1.r4.pgf
meta-probeset file: MoGene-1_1-st-v1.r4.mps

label: Biotin

organism: Mus musculus

source_name: bone marrow derived macrophage

label_protocol: Replicate 250 ng samples of total RNA derived from two separate wells per time point were enzymatically fragmented and biotinylated using the Ambion Whole Transcript Expression Kit (Ambion)

growth_protocol: Bone marrow derived macrophages were prepared from femurs of 7-8 week old male BALB/c or C57BL/6 mice

extract_protocol: Total RNA was harvested from the cells using an RNeasy Plus kit (Qiagen)

treatment_protocol: On day 6 cells were harvested, counted, re-suspended in complete medium with 10,000 U/ml CSF1 and seeded into 24-well tissue culture plates at a density of 200,000 cells/well.  24 h later (day 7) cells were treated with any of LPS, interferon-beta or interferon-gamma

tag: background strain

tag: treatment type

value: LPS

tag: treatment dose

value: 0.5 ng/ml

tag: time post treatment

value: 24 hours


title: Mesenchymal_A_rep1

description:
Sample name: A1
SDF file: 6898481097.sdf
Metrics file: 6898481097Metrics.txt
Annotation file: annotation_6898481097.csv

value: alfa-MEM

value: FTS

value: FALSE

value: A


title: 4h-0.5ng-LPS-2_C57BL6

description: 4h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 2

value: 4 hours


title: yFR399vsyFR116_Gal11-9MYCvsUntagged_30minRapa_Rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged strain (yFR399) versus the ChIP in untagged strain (yFR116), after 30 min with 200ng/ml rapamycin.


title: yFR116_WT_Rpb3vsInput_noRapa_Rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells.


title: yFR116_WT_Rpb3vsInput_noRapa_Rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells.


title: 4h-0.5ng-LPS-1_C57BL6

description: 4h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 1


title: 8h-0.5ng-LPS-1_C57BL6

description: 8h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 1

value: 8 hours


title: 2h-0.5ng-LPS-2_C57BL6

description: 2h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 2

value: 2 hours


title: yFR116_WT_Rpb3vsInput_30minRapa_Rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells, after 30 min with 200ng/ml rapamycin.

treatment_protocol:
Rapamycin-30min-200ng/ml
Rpb3 W0012 antibody is from neoclone). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009.

treatment_protocol:
Rapamycin-30min-200ng/ml
null


title: 8h-0.5ng-LPS-2_C57BL6

description: 8h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 2


title: 2h-0.5ng-LPS-1_C57BL6

description: 2h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 1


title: 1h-0.5ng-LPS-1_C57BL6

description: 1h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 1

value: 1 hour


title: yFR1651vsyFR1526_ExoCTDS7A-FLAG_Gal11-18MYCvsUntagged_90minRapa_rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S7A-FLAG strain (yFR1651) versus the ChIP in untagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S7A-FLAG strain (yFR1526), after 90 min with 1µg/ml rapamycin.

growth_protocol: YNB-HIS_Glu_90minRapamycin. YNB-HIS + 2%Glucose is inoculated at OD600=0.1 from an overnight preculture in the appropriate media and allowed to grow until it reaches OD600=0.5-0.6. Cell cultures are treated with 1 microg/ml of Rapamycin and allowed to grow for additional 90 min. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transferred to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

treatment_protocol:
Rapamycin-90min-1µg/ml
9E10 anti-Myc antibody (home-prepared). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65'C to reverse the crosslinks. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.


title: yFR117_WT_MockIgGvsInput_rep1

description: Biological replicate 1 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.

growth_protocol: Standard_YEPD_50ml. 50mL YEP+2% Glucose is inoculated at OD60= 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reach OD600=0.6-1.0. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde (optional). Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

treatment_protocol: Rabbit polyclonal antibody from Enzo Life Sciences. Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Antibody is   added (5uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL of cell lysate are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.

value: rabbit IgG


title: yFR1651vsyFR1526_ExoCTDS7A-FLAG_Gal11-18MYCvsUntagged_90minRapa_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S7A-FLAG strain (yFR1651) versus the ChIP in untagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S7A-FLAG strain (yFR1526), after 90 min with 1µg/ml rapamycin.


title: yFR1645vsyFR1499_ExoCTDwt-FLAG_Gal11-18MYCvsUntagged_90minRapa_rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG strain (yFR1645) versus the ChIP in untagged endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG strain (yFR1499), after 90 min with 1µg/ml rapamycin.


title: yFR117_WT_MockIgGvsInput_rep2

description: Biological replicate 2 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR117 (WT) cells.


title: 24h_5ng-LPS-2_C57BL6

description: 24h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 2

value: 5 ng/ml


title: 1h-0.5ng-LPS-2_C57BL6

description: 1h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 2


title: 8h_5ng-LPS-2_C57BL6

description: 8h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 2


title: 24h_5ng-LPS-1_C57BL6

description: 24h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 1


title: yFR116_WT_Rpb3vsInput_30minRapa_Rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR116 (WT) cells, after 30 min with 200ng/ml rapamycin.


title: yFR1526_exoCTD-S7A-FLAGvsInput_90minRapa_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTD-S7A-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1526 (endogenous Rpb1-FRB, exogenous Rpb1-CTD-S7A-FLAG mutant) cells, after 90 min with 1µg/ml rapamycin.

treatment_protocol:
Rapamycin-90min-1µg/ml
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with PBS + 0.5% BSA (freshly made), and resuspended in PBS + 0.5% BSA. Anti-Flag M2 (Sigma F3165) is added (5ug/ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody is added to 500 uL of lysate, and incubated overnight at 4'C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: Flag M2 R (Sigma)

treatment_protocol:
Rapamycin-90min-1µg/ml
null


title: yFR1499_exoCTDwt-FLAGvsInput_90minRapa_rep3

description: Biological replicate 1 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTDwt-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1649vsyFR1503_ExoCTDS5A-FLAG_Gal11-18MYCvsUntagged_90minRapa_rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S5A-FLAG strain (yFR1649) versus the ChIP in untagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S5A-FLAG strain (yFR1503), after 90 min with 1µg/ml rapamycin.

value: 9E10 (lab stock by AF)


title: yFR1645vsyFR1499_ExoCTDwt-FLAG_Gal11-18MYCvsUntagged_90minRapa_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG strain (yFR1645) versus the ChIP in untagged endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG strain (yFR1499), after 90 min with 1µg/ml rapamycin.


title: yFR1499_exoCTDwt-FLAGvsInput_90minRapa_rep4

description: Biological replicate 2 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTDwt-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1649vsyFR1503_ExoCTDS5A-FLAG_Gal11-18MYCvsUntagged_90minRapa_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S5A-FLAG strain (yFR1649) versus the ChIP in untagged endogenous Rpb1-FRB, exogenous Rpb1-CTD-S5A-FLAG strain (yFR1503), after 90 min with 1µg/ml rapamycin.


title: 1h_5ng-LPS-2_C57BL6

description: 1h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 2


title: 4h_5ng-LPS-1_C57BL6

description: 4h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 1


title: 4h_5ng-LPS-2_C57BL6

description: 4h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 2


title: 8h_50ng-LPS-1_C57BL6

description: 8h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 1

value: 50 ng/ml


title: 2h_5ng-LPS-1_C57BL6

description: 2h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 1


title: yFR1526_exoCTD-S7A-FLAGvsInput_90minRapa_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTD-S7A-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1526 (endogenous Rpb1-FRB, exogenous Rpb1-CTD-S7A-FLAG mutant) cells, after 90 min with 1µg/ml rapamycin.


title: 1h_5ng-LPS-1_C57BL6

description: 1h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 1


title: 8h_50ng-LPS-2_C57BL6

description: 8h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 2


title: 8h_5ng-LPS-1_C57BL6

description: 8h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 1


title: 24h_50ng-LPS-2_C57BL6

description: 24h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 2


title: yFR1499_MockvsInput_noRapamycin_rep2

description: Biological replicate 2 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells.

growth_protocol: YNB-HIS_Glu. YNB-HIS + 2%Glucose is inoculated at OD600=0.1 from an overnight preculture in the appropriate media and allowed to grow until it reaches OD600=0.8. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transferred to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

treatment_protocol: Normal rabbit IgG, a polyclonal antibody that is routinely used as a non-specific IgG Mock control in ChIP, is from Millipore (#12-370). Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Antibody is added (2ug / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL Pepstatin.


title: 24h-0.5ng-LPS-1_C57BL6

description: 24h 0.5ng/ml LPS treated macrophage C0.57BL/6 derived replicate 1


title: 2h_50ng-LPS-2_C57BL6

description: 2h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 2


title: 2h_5ng-LPS-2_C57BL6

description: 2h 5ng/ml LPS treated macrophage C57BL/6 derived replicate 2


title: 4h_50ng-LPS-2_C57BL6

description: 4h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 2


title: yFR1499_exoCTDwt-FLAGvsInput_90minRapa_20131216_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTDwt-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.


title: 4h_50ng-LPS-1_C57BL6

description: 4h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 1


title: 24h_50ng-LPS-1_C57BL6

description: 24h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 1


title: yFR1499_exoCTDwt-FLAGvsInput_90minRapa_20131216_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTDwt-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1499_MockvsInput_90minRapamycin_rep2

description: Biological replicate 2 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.

treatment_protocol:
Rapamycin-90min-1µg/ml
Normal rabbit IgG, a polyclonal antibody that is routinely used as a non-specific IgG Mock control in ChIP, is from Millipore (#12-370). Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Antibody is added (2ug / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL Pepstatin.


title: yFR1503_exoCTD-S5A-FLAGvsInput_90minRapa_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTD-S5A-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1503 (endogenous Rpb1-FRB, exogenous Rpb1-CTD-S5A-FLAG mutant) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1499_endoRPB1-FRBvsInput_noRapa_20131216_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using the anti-FRB antibody which detects endogenous Rpb1-FRB tagged subunit, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells.

value: mTOR (human FRB domain)_Anchor Away tag


title: 2h_50ng-LPS-1_C57BL6

description: 2h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 1

value: LPS


title: yFR1503_exoCTD-S5A-FLAGvsInput_90minRapa_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTD-S5A-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1503 (endogenous Rpb1-FRB, exogenous Rpb1-CTD-S5A-FLAG mutant) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1499_MockvsInput_90minRapamycin_rep1

description: Biological replicate 1 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1499_exoCTDwt-FLAGvsInput_noRapa_20131216_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTDwt-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells.

treatment_protocol: Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with PBS + 0.5% BSA (freshly made), and resuspended in PBS + 0.5% BSA. Anti-Flag M2 (Sigma F3165) is added (5ug/ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice (with PBS/BSA or Lysis buffer), and resuspended in 30uL (PBS/BSA or Lysis buffer) / ChIP. 30uL of resuspended beads/antibody is added to 500 uL of lysate, and incubated overnight at 4'C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.


title: yFR1499_MockvsInput_noRapamycin_rep1

description: Biological replicate 1 of 2. Mock IgG occupancy, using the rabbit anti-IgG, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells.


title: yFR1499_exoCTDwt-FLAGvsInput_noRapa_20131216_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using the anti-FLAG antibody which detects exogenous Rpb1-CTDwt-FLAG tagged, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells.


title: yFR1499_endoRPB1-FRBvsInput_90minRapa_20131216_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using the anti-FRB antibody which detects endogenous Rpb1-FRB tagged subunit, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.

treatment_protocol:
Rapamycin-90min-1µg/ml
Rabbit polyclonal antibody from Enzo Life Sciences. Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. Antibody is   added (5uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. 500uL of cell lysate are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.


title: yFR1499_endoRPB1-FRBvsInput_noRapa_20131216_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using the anti-FRB antibody which detects endogenous Rpb1-FRB tagged subunit, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells.


title: yFR1499_endoRPB1-FRBvsInput_90minRapa_20131216_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using the anti-FRB antibody which detects endogenous Rpb1-FRB tagged subunit, measured by the ratio of ChIP versus Input DNA in yFR1499 (endogenous Rpb1-FRB, exogenous Rpb1-CTDwt-FLAG) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1638vsyFR763-kin28as_Gal11-9xMYCvsUntagged_rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged WT strain (yFR1636) versus the ChIP in untagged WT strain (yFR762), after 15 min with 6uM NAPP1.

growth_protocol: YEP-Glu_NAPP1. YEP+2% Glucose is inoculated at OD600=0.1 from an overnight preculture in the appropriate media and allowed to grow until it reach OD600=0.6-0.8. NA-PP1 is added at final concentration 6uM and cultures allowed to grow for an additional 15 min. Culture is poured/divided in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

treatment_protocol:
6uM NAPP1_15 min
9E10 anti-Myc antibody (home-prepared). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65'C to reverse the crosslinks. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.


title: 24h_5ng-LPS-2_BALBc

description: 24h 5ng/ml LPS treated macrophage BALB/c derived replicate 2

value: BALB/c


title: 4h_5ng-LPS-2_BALBc

description: 4h 5ng/ml LPS treated macrophage BALB/c derived replicate 2


title: yFR1638vsyFR763-kin28as_Gal11-9xMYCvsUntagged_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged kin28-as strain (yFR1638) versus the ChIP in untagged kin28-as strain (yFR763), after 15 min with 6uM NAPP1.


title: yFR1636vsyFR762-WT_Gal11-9xMYCvsUntagged_rep2


title: 1h_50ng-LPS-2_C57BL6

description: 1h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 2


title: 1h_50ng-LPS-1_C57BL6

description: 1h 50ng/ml LPS treated macrophage C507BL/6 derived replicate 1


title: 0h_Con-1_C57BL6

description: 0h Control C57BL/6 derived macrophage replicate 1

value: None -Control

value: 0 hours


title: 2h_5ng-LPS-2_BALBc

description: 2h 5ng/ml LPS treated macrophage BALB/c derived replicate 2


title: 1h_5ng-LPS-2_BALBc

description: 1h 5ng/ml LPS treated macrophage BALB/c derived replicate 2


title: 0h_Con-2_C57BL6

description: 0h Control C57BL/6 derived macrophage replicate 2


title: 24h_Ifng-2_BALBc

description: 24h IFN-gamma treated macrophage BALB/c derived replicate 2

value: Interferon-gamma

value: 10 U/ml

value: 24 hours


title: 8h_5ng-LPS-2_BALBc

description: 8h 5ng/ml LPS treated macrophage BALB/c derived replicate 2


title: 8h_Ifng-2_BALBc

description: 8h IFN-gamma treated macrophage BALB/c derived replicate 2


title: 4h_5ng-LPS-1_BALBc

description: 4h 5ng/ml LPS treated macrophage BALB/c derived replicate 1

value: 4 hours


title: 1h_5ng-LPS-1_BALBc

description: 1h 5ng/ml LPS treated macrophage BALB/c derived replicate 1


title: 24h_5ng-LPS-1_BALBc

description: 24h 5ng/ml LPS treated macrophage BALB/c derived replicate 1


title: 2h_5ng-LPS-1_BALBc

description: 2h 5ng/ml LPS treated macrophage BALB/c derived replicate 1


title: yFR763_kin28as_Rpb3vsInput_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR763 (kin28-as mutant) cells, after 15 min with 6uM NAPP1.

treatment_protocol:
6uM NAPP1_15 min
Rpb3 W0012 antibody is from neoclone). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 5mL PBS + 0.5% BSA. W0012 antibody is added (3uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed in cold room (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA. Ref: Bataille A.R., Robert F. Methods in Enzymology 2009.

value: Rpb3 (W0012 neoclone)

treatment_protocol:
6uM NAPP1_15 min
null


title: yFR1636vsyFR762-WT_Gal11-9xMYCvsUntagged_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged WT strain (yFR1636) versus the ChIP in untagged WT strain (yFR762), after 15 min with 6uM NAPP1.


title: 24h_Ifng-1_BALBc

description: 24h IFN-gamma treated macrophage BALB/c derived replicate 1


title: yFR762_WT-KIN28_Rpb3vsInput_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR762 (WT for yFR763) cells, after 15 min with 6uM NAPP1.


title: yFR763_kin28as_Rpb3vsInput_rep2

description: Biological replicate 2 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR763 (kin28-as mutant) cells, after 15 min with 6uM NAPP1.


title: WT pellet vs WT supernatant (1 of 2)

city: PARIS

first: sophie

last: lemoine

address: 46 rue d'Ulm

department: Biologie

country: France

institute: Ecole Normale Supérieure

description: Biological replicate 1 of 2

hyb_protocol: 2µg of labelled cDNA is mixed with one volume of 2X GE buffer (Agilent) and 10% of blocking agent. The hybridization sample volume is dispense on the selected microarray and incubated overnight at 65°C in a hybridization oven (Agilent). The array slides are washed 1 minute in "wash 1 buffer" at room temperature and one minute in "wash 2 buffer" at 37°C. The array slides are then air dryed and ready for scanning

scan_protocol: Scanned on a genepix 4000B scanner (Axon)

data_processing:
Scanning software: GenePix Pro 6.1
Normalization using Goulphar WebTool http://transcriptome.ens.fr/goulphar/
Genepix flags are removed from the analysis. Spots are considered as saturating spot above 60000 and removed. Background is not subtracted from the main signal. Global median normalisation method

source_name: WT pellet (P18k)

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described (Koszul et al., 2004). We used 0.11 μg of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy5-dCTP.

growth_protocol: Cells were grown in a YPDA at 25°C to a final OD of 0.8-1.0 and subsequently shifted to 37°C for 15 minutes by the addition of an equal volume of pre-warmed medium.

extract_protocol: DNA extraction using a QIAGEN kit

treatment_protocol: Experiments were performed according to previously published methods (Assenholt et al., 2008; Rougemaille et al., 2008)

tag: genotype

source_name: WT supernatant (SN2k)

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described in previously published methods (Koszul et al., 2004). We used 1.1 μg of of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy3-dCTP.


title: yFR762_WT-KIN28_Rpb3vsInput_rep1

description: Biological replicate 1 of 2. RNAPII occupancy, using anti-Rpb3 subunit antibody, measured by the ratio of ChIP versus Input DNA in yFR762 (WT for yFR763) cells, after 15 min with 6uM NAPP1.


title: 8h_5ng-LPS-1_BALBc

description: 8h 5ng/ml LPS treated macrophage BALB/c derived replicate 1

value: 8 hours


title: WT pellet vs WT supernatant (2 of 2)

description: Biological replicate 2 of 2

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described (Koszul et al., 2004). We used 0.12 μg of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy3-dCTP.

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described in previously published methods (Koszul et al., 2004). We used 0.95 μg of of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy5-dCTP.


title: mft1 delta pellet vs mft1 delta supernatant (2 of 2)

description: Biological replicate 2 of 2

source_name: mft1 delta pellet (P18k)

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described (Koszul et al., 2004). We used 0.115 μg of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy3-dCTP.

source_name: mft1 delta supernatant (SN2k)

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described in previously published methods (Koszul et al., 2004). We used 0.975 μg of of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy5-dCTP.


title: mft1 delta pellet vs mft1 delta supernatant (1 of 2)

description: Biological replicate 1 of 2

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described (Koszul et al., 2004). We used 0.114 μg of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy5-dCTP.

label_protocol: DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described in previously published methods (Koszul et al., 2004). We used 1.15 μg of of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy3-dCTP.


title: 2h_Ifng-2_BALBc

description: 2h IFN-gamma treated macrophage BALB/c derived replicate 2

value: 2 hours


title: 1h_Ifng-2_BALBc

description: 1h IFN-gamma treated macrophage BALB/c derived replicate 2

value: 1 hour


title: yFR1544_Srb4-FRB_rapamycin_TFIIBvsInput_rep1

description: Biological replicate 1 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR1544 (Srb4-FRB tagged) cells, after 90 min with 1µg/ml rapamycin.

growth_protocol: YEP-Glu_Rapamycin-90min-1µg/ml. YEP+2%Glucose is inoculated at OD600=0.1 from an overnight preculture in the appropriate media and allowed to grow until it reachs OD600=0.5-0.6. Cell cultures are treated with 1 microg/ml of Rapamycin and allowed to grow for additional 90 min. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min.  Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde. Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

treatment_protocol:
Rapamycin-90min-1µg/ml
Anti-yTFIIB (from Richard Young) antibody. Protein G Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 2mL PBS + 0.5% BSA. Anti-yTFIIB (from Richard Young) antibody is added (2uL / ChIP), and incubated overnight at 4C in haematological mixer. Beads are washed twice with Lysis buffer, and resuspended in 30uL Lysis buffer / ChIP. 30uL of resuspended beads/antibody is added to 400-600 uL of lysate, and incubated overnight at 4C in haematological mixer. Beads are then washed using the Dynal magnet system twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl, twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting after a brief centrifugation at 4'C, and 50uL TE + 1% SDS is added for elution. Beads are incubated overnight at 65'C for elution and reverse crosslink. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 50uL TE + 1% SDS to 5uL WCE, and put overnight at 65'C to reverse the crosslinks with the ChIP samples. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate Wash Buffer: 10mM Tris-HCl, pH 8.0 250mM LiCl 0.5% NP40 0.5% Na-deoxycholate 1mM EDTA.

value: TFIIB


title: 4h_Ifng-1_BALBc

description: 4h IFN-gamma treated macrophage BALB/c derived replicate 1


title: 4h_Ifng-2_BALBc

description: 4h IFN-gamma treated macrophage BALB/c derived replicate 2


title: 24h_Ifnb-2_BALBc

description: 24h IFN-beta treated macrophage BALB/c derived replicate 2

value: Interferon-beta


title: 4h_Ifnb-2_BALBc

description: 4h IFN-beta treated macrophage BALB/c derived replicate 2


title: yFR1321_WT_rapamycin_TFIIBvsInput_rep2

description: Biological replicate 2 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR1321 (WT for yFR1325 and yFR1544) cells, after 90 min with 1µg/ml rapamycin.


title: 2h_Ifnb-1_BALBc

description: 2h IFN-beta treated macrophage BALB/c derived replicate 1


title: 8h_Ifng-1_BALBc

description: 8h IFN-gamma treated macrophage BALB/c derived replicate 1


title: 2h_Ifng-1_BALBc

description: 2h IFN-gamma treated macrophage BALB/c derived replicate 1


title: 2h_Ifnb-2_BALBc

description: 2h IFN-beta treated macrophage BALB/c derived replicate 2


title: 24h_Ifnb-1_BALBc

description: 24h IFN-beta treated macrophage BALB/c derived replicate 1


title: 8h_Ifnb-2_BALBc

description: 8h IFN-beta treated macrophage BALB/c derived replicate 2


title: 1h_Ifng-1_BALBc

description: 1h IFN-gamma treated macrophage BALB/c derived replicate 1


title: yFR1325_Srb5-FRB_rapamycin_TFIIBvsInput_rep2

description: Biological replicate 2 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR1325 (Srb5-FRB tagged) cells, after 90 min with 1µg/ml rapamycin.


title: 4h_Ifnb-1_BALBc

description: 4h IFN-beta treated macrophage BALB/c derived replicate 1


title: yFR1325_Srb5-FRB_rapamycin_TFIIBvsInput_rep1

description: Biological replicate 1 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR1325 (Srb5-FRB tagged) cells, after 90 min with 1µg/ml rapamycin.


title: 8h_Ifnb-1_BALBc

description: 8h IFN-beta treated macrophage BALB/c derived replicate 1


title: 1h_Ifnb-2_BALBc

description: 1h IFN-beta treated macrophage BALB/c derived replicate 2


title: yFR399vsyFR116_9Myc-Gal11-Med15vsUntagged_rep2

description: Biological replicate 2 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged strain (yFR399) versus the ChIP in untagged strain (yFR116).

growth_protocol: Standard_YEPD_50mL. 50mL of YEP+2% Glucose is inoculated at OD600= 0.1 from an overnight preculture in the appropriate media and allowed to grow until it reach OD600= 0.6-1.0. Culture is poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde for crosslinking, and incubate on rotating wheel for 30 min. Crosslinking is stopped by addition of 2.5mL 2.5M Glycine to quench formaldehyde (optional). Crosslinked cells are pelleted at 4'C, 3000 rpm, resuspended in 40mL ice cold TBS and pelleted again. This wash is repeated a second time. Cell pellets are resuspended in the remaining liquid (~1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells pellet snap-freeze in liquid nitrogen and stock at -80'C.

treatment_protocol: 9E10 anti-Myc antibody (home-prepared). Anti-mouse Dynabeads (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL PBS + 0.5% BSA. 9E10 anti-Myc antibody (home-prepared)  is added (10uL / ChIP), and incubated overnight at 4'C in haematological mixer. Beads are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 30uL PBS + 0.5% BSA / ChIP. A mix of 250uL of cell lysate + 250uL of Lysis Buffer containing protease inhibitors are added to 30uL beads, and incubated overnight at 4'C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65'C to reverse the crosslinks. Lysis buffer: 50mM HEPES-KOH, pH 7.5 140mM NaCl 1mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 1mM PMSF 1mM Benzamidine 10ug/mL Aprotinin 1ug/mL Leupeptin 1ug/mL. Pepstatin.

value: 9E10 (lab stock by AF)


title: yFR1544_Srb4-FRB_rapamycin_TFIIBvsInput_rep2

description: Biological replicate 2 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR1544 (Srb4-FRB tagged) cells, after 90 min with 1µg/ml rapamycin.


title: yFR1321_WT_rapamycin_TFIIBvsInput_rep1

description: Biological replicate 1 of 2. TFIIB occupancy, using anti-TFIIB antibody, measured by the ratio of ChIP versus Input DNA in yFR1321 (WT for yFR1325 and yFR1544) cells, after 90 min with 1µg/ml rapamycin.


title: Tumor 4

city: Badalona

first: Miguel

last: Torres-Martin

state: Barcelona

address: Carretera de Canyet, s/n

department: Clinical Genomics Unit

country: Spain

institute: Hospital Universitari Germans Trias i Pujol

hyb_protocol: The entire labeled RNA was hybridized on a microarray for 16 hours of hybridization at 45ºC, washing and staining of microarrays was performed using a Fluidics Station 450 (Affymetrix, Santa Clara, CA). Hybridization, washing and scanning was performed according to manufacturer's recommendations.

scan_protocol: GeneChips were scanned in a GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA)

data_processing: CEL files were generated from DAT files using GCOS software (Affymetrix). Normalized expression signals were calculated from Affymetrix CEL files using RMA (Irizarry et al., 2003)

source_name: Tumor specimen

label_protocol: 500 ng total RNA was labeled using the FlashTag Biotin HSR Kit (Genisphere) according to manufacturer's instructions.

extract_protocol: Total RNA was extracted with Ambion mirVanaTM miRNA Isolation Kit (CA, USA) according to the manufacturer's instructions.

treatment_protocol: Samples were frozen in liquid Nitrogen after surgical extraction and keeped at -80 until total RNA purification

tag: tissue

value: Vestibular Schwannoma

tag: batch


title: Tumor 3


title: Tumor 5


title: yFR399vsyFR116_9Myc-Gal11-Med15vsUntagged_rep1

description: Biological replicate 1 of 2. Mediator Gal11-Med15-9MYC tagged subunit occupancy, using the anti-MYC antibody, measured by the ratio of ChIP in tagged strain (yFR399) versus the ChIP in untagged strain (yFR116).

tag: strain


title: Tumor 11


title: Tumor 10


title: Tumor 9


title: Tumor 2


title: 0h_Con-1_BALBc

description: 0h Control BALB/c derived macrophage replicate 1

treatment_protocol: On day 6 cells were harvested, counted, re-suspended in complete medium with 10,000 U/ml CSF1 and seeded into 24-well tissue culture plates at a density of 200,000 cells/well.  24 h later (day 7) cells were treated with any of LPS, interferon-beta or interferon-gamma