title: 435kinv.A4.L (repli-seq)


title: 435kinv.A4.E (repli-seq)

status: Public on Jan 21 2019

type: SRA

city: Tallahassee

first: David

middle: M

last: Gilbert

state: FL

address: 319 Stadium Drive

department: Biology

country: USA

institute: Florida State University

library_source: genomic

data_processing:
Reads are mapped to mm10 using bowtie2.
Coverage data are calculated from mapped reads using samtools and bedtools
Log2 ratio of early to late coverage is calculated in 5kb windows
Log 2 ratio data were IQR normalized to 4.59
Genome_build: mm10
Supplementary_files_format_and_content: All .txt datasets are normalized to RPM for both early and late fractions before taking log2 ratios, and then Loess smoothed

molecule: genomic DNA

organism: Mus musculus

growth_protocol: Mouse ESCs were maintained on 1% gelatin coated dishes. 46C was grown in GMEM supplemented with 10% FBS and LIF; hybrid cells (Cas/129 and V6.5) in DMEM supplemented with 2i plus LIF.

extract_protocol:
Genome-wide RT profiles were constructed as previously described. Briefly, cells were pulse labeled with BrdU for 2 hours and separated into early and late S-phase fractions by Propidium iodide staining and flow cytometry. BrdU-substituted DNA from 40k early and late S-phase cells was immunoprecipitated by anti-BrdU antibody (Becton Dickinson 347580)
Sequencing libraries were prepared by NEBNext Ultra DNA Library Prep Kit for Illumina (E7370).

treatment_protocol: CRISPR-mediated deletion or inversions

tag: cell type

value: mESC

tag: hybrid cells

tag: s phase fraction

value: Early S fraction

tag: protocol


title: 46C.E (repli-seq)


title: 680kinv.B10.L (repli-seq)

value: Late S fraction


title: 680kinv.80.E (repli-seq)


title: 680kinv.C5.E (repli-seq)


title: 680kinv.80.L (repli-seq)


title: 28.5kInv.B7.rep2.E (repli-seq)


title: 680kinv.B10.E (repli-seq)


title: 28.5kInv.B7.rep2.L (repli-seq)


title: 680kinv.C5.L (repli-seq)


title: 28.5kinv.B7.rep1.E (repli-seq)


title: 350kinv.L (repli-seq)


title: 45kinv.rep2.L (repli-seq)


title: 350kinv.E (repli-seq)


title: 45kinv.rep2.E (repli-seq)


title: 28.5kinv.B7.rep1.L (repli-seq)


title: ac.del.E4.rep2.L (repli-seq)


title: 4k.b.del.B4.L (repli-seq)


title: 4k.b.c.del.G12.E (repli-seq)


title: 45kinv.rep1.E (repli-seq)


title: 4k.b.c.del.G12.L (repli-seq)


title: 4k.b.del.B4.E (repli-seq)


title: 45kinv.rep1.L (repli-seq)


title: 4k.b.del.B1.L (repli-seq)


title: bc.del.B3.E (repli-seq)


title: ab.del.D12.L (repli-seq)


title: 4k.b.del.B1.E (repli-seq)


title: 24.5kinv.3.E (repli-seq)


title: bc.del.B3.L (repli-seq)


title: c.del.C9.E (repli-seq)


title: ab.del.D10.E (repli-seq)


title: c.del.C6.L (repli-seq)


title: ab.del.D12.E (repli-seq)


title: c.del.C9.L (repli-seq)


title: ab.del.D10.L (repli-seq)


title: c.del.B11.E (repli-seq)


title: c.del.C6.E (repli-seq)


title: c.del.B11.L (repli-seq)


title: c.del.A12.E (repli-seq)


title: b.del.D6.L (repli-seq)


title: b.del.D6.E (repli-seq)


title: b.del.D10.L (repli-seq)


title: a.del.53.rep2.E (repli-seq)


title: b.del.D10.E (repli-seq)


title: a.del.53.E (repli-seq)


title: abc.del.E2.E (repli-seq)


title: a.del.53.rep2.L (repli-seq)


title: abc.del.E2.L (repli-seq)


title: a.del.53.L (repli-seq)


title: abc.del.A6.rep2.E (repli-seq)


title: abc.del.A6.rep2.L (repli-seq)


title: 28kdel.9.E (repli-seq)


title: 28.5kdel.A10.rep2.L (repli-seq)


title: 28kdel.9.L (repli-seq)


title: abc.del.A6.rep1.E (repli-seq)


title: 28.5kdel.A10.rep1.E (repli-seq)


title: 4k.del.G4.E (repli-seq)


title: 28.5kdel.A10.rep1.L (repli-seq)


title: 4kdel.C6.rep2.L (repli-seq)


title: 4kdel.C6.rep2.E (repli-seq)


title: 4kdel.C6.rep1.L (repli-seq)


title: 4kdel.C6.rep1.E (repli-seq)


title: 18kdel.H5.L (repli-seq)


title: 14kdel.D11.E (repli-seq)


title: 18kdel.D6.L (repli-seq)


title: 18kdel.E12.E (repli-seq)


title: 18kdel.E12.L (repli-seq)


title: 18kdel.H5.E (repli-seq)


title: abc.del.A6.rep1.L (repli-seq)


title: 29kdel.D8.E (repli-seq)


title: 29kdel.D8.L (repli-seq)


title: 245kdel.28.E (repli-seq)


title: 245kdel.25.L (repli-seq)


title: 245kdel.25.E (repli-seq)


title: 245kdel.28.L (repli-seq)


title: c.del.A12.L (repli-seq)


title: 245kdel.F9.L (repli-seq)


title: 28.5kdel.A10.rep2.E (repli-seq)


title: 4k.del.G4.L (repli-seq)


title: 245kdel.F9.E (repli-seq)


title: 100kdel.C3.NPC.E (repli-seq)


title: 100kdel.C6.L (repli-seq)


title: 100kdel.C6.E (repli-seq)


title: 100kdel.C3.rep2.E (repli-seq)


title: 100kdel.C3.rep2.L (repli-seq)


title: 100kdel.C3.NPC.L (repli-seq)


title: 14kdel.D11.L (repli-seq)


title: 100kdel.C3.rep1.E (repli-seq)


title: 100kdel.C3.rep1.L (repli-seq)


title: 100kdel.L (repli-seq)


title: 100kdel.B6.E (repli-seq)


title: 100kdel.B6.L (repli-seq)


title: 100kdel.E (repli-seq)


title: 18kdel.D6.E (repli-seq)


title: 345kdel.E (repli-seq)


title: 680kdel.16.L (repli-seq)


title: 680kdel.79.L (repli-seq)


title: 680kdel.79.E (repli-seq)


title: 680kdel.13.E (repli-seq)


title: 680kdel.13.L (repli-seq)


title: 680kdel.16.E (repli-seq)


title: 145kdel.G5.L (repli-seq)


title: 345kdel.L (repli-seq)


title: 435kdel.L (repli-seq)


title: 145kdel.G5.E (repli-seq)


title: 435kdel.E (repli-seq)


title: 435kdel.B8.L (repli-seq)


title: 145k.30kdel.E12.L (repli-seq)


title: 435kdel.B8.E (repli-seq)


title: 30kdel.C2.L (repli-seq)


title: 30kdel.B11.E (repli-seq)


title: 30kdel.C2.E (repli-seq)


title: 500kdel.L (repli-seq)


title: 24.5kinv.3.L (repli-seq)


title: 350kdel.L (repli-seq)


title: 350kdel.E (repli-seq)


title: 145k.30kdel.E12.E (repli-seq)


title: 45kdel.E (repli-seq)


title: 145k.30kdel.A3.E (repli-seq)


title: 2kdel.E (repli-seq)


title: 145k.30kdel.A3.L (repli-seq)


title: 2kdel.L (repli-seq)


title: 30kdel.B11.L (repli-seq)


title: 45kdel.L (repli-seq)


title: 500kdel.E (repli-seq)


title: sdg8 seedlings Replicate 2

type: RNA

city: Strasbourg

first: Wen-Hui

last: Shen

address: 12 rue du General Zimmer

country: France

institute: IBMP-CNRS

description:
sdg8-2
Biological replicate 2 of sdg8 seedlings.

hyb_protocol: Hybridization Oven 645 at 45°C 16h, wash Fluidics Station 450.

scan_protocol: GeneChip Scanner 3000, Command Console Software 4.0.

data_processing: Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Affy packages in R.

label: biotin

molecule: total RNA

organism: Arabidopsis thaliana

label_protocol: Affymetrix GeneChip 3' IVT PLUS Reagent Kit.

growth_protocol: Plants were grown in growth chamber under 16h light and 8h dark at 22°C.

extract_protocol: 2 µg of total RNA were labeled according to Agilent's instructions. Please see manufacturer’s web site at http://www.agilent.com/.

treatment_protocol: Total RNA was extracted using the Trizol reagent (Invitrogen) followed by RNeasy mini kit (Qiagen) clean-up according to manufacturer's instructions.

tag: ecotype

value: Columbia

tag: genotype/variation

tag: age

value: 6 days

tag: tissue

value: Seedlings


title: sdg8 seedlings Replicate 1

description:
sdg8-1
Biological replicate 1 of sdg8 seedlings.


title: Wild-type seedlings Replicate 3

description:
Col-3
Biological replicate 3 of wild-type seedlings.

value: Wild-type


title: hub2 seedlings Replicate 1

description:
hub2-1
Biological replicate 1 of hub2 seedlings.


title: sdg8hub2 seedlings Replicate 3

description:
sdg8hub2-3
Biological replicate 3 of sdg8hub2 seedlings.


title: sdg8 seedlings Replicate 3

description:
sdg8-3
Biological replicate 3 of sdg8 seedlings.


title: sdg8hub2 seedlings Replicate 2

description:
sdg8hub2-2
Biological replicate 2 of sdg8hub2 seedlings.


title: hub2 seedlings Replicate 2

description:
hub2-2
Biological replicate 2 of hub2 seedlings.


title: Wild-type seedlings Replicate 2

description:
Col-2
Biological replicate 2 of wild-type seedlings.


title: sdg8hub2 seedlings Replicate 1

description:
sdg8hub2-1
Biological replicate 1 of sdg8hub2 seedlings.


title: Wild-type seedlings Replicate 1

description:
Col-1
Biological replicate 1 of wild-type seedlings.


title: hub2 seedlings Replicate 3

description:
hub2-3
Biological replicate 3 of hub2 seedlings.


title: yFR1669_MYCvsInput_CTDwt-Endo-rep2

type: genomic

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

country: Canada

institute: IRCM

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1669_MYCvsInput_CTDwt-Endo-rep1-2_20140220__S.cerevisiaeApr11_448927.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

hyb_protocol: Combined samples were resuspended in 110 µL of a hybridization mix (5 µL of 10 mg/mL ssDNA, 5 µL of 8 mg/mL tRNA and 100 µL DIG EASY (Roche, 11603558001)) and incubated 5 min at 95˚C followed by 5 min at 42˚C. Samples were incubated overnight on Agilent microarrays in SureHyb chambers with rotation. Microarrays were washed 5 min at room temperature with Wash I buffer (6X SSPE, 0.005% N-lauroylsarcosine) and washed a second time with a pre-warmed (42˚C) Wash II buffer (0.06X SSPE) during 5 min at room temperature.

scan_protocol: Microarrays were scanned using an InnoScan900 (Innopsys) at 2µm resolution. Scanner was set as follow: pixel size = 2µm; speed = 20; Scan Mode = Normal; Acquisition Mode = Simultaneous; Focus = Auto. Detection gain for 635 and 532 wavelengths were manually adjusted in order to have less than 5% spot saturation.

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

organism: Saccharomyces cerevisiae

label_protocol: DNA blunting was done starting from 40 µL of ChIP DNA or about 400 ng of Input DNA. Samples were added to 11 µL 10X NEB2 buffer, 0.5 µL 10 mg/mL BSA, 0.5 µL 20 mM dNTPs and 0.2 µL (0.6 units) of T4 DNA pol (New England Biolabs, M0203L) in a final volume of 70 µL and incubated 20 min at 12˚C. Then, 11.5 µL of 3 M NaOAc pH 5.2 and 2 µL of 20 mg/mL glycogen were added, followed by a phenol/chloroform/isoamyl alcool extraction ethanol precipitation. DNA pellets were resuspended in 25 µL H2O and DNA linkers were ligated using 10 µL 5x ligase buffer, 6.7 µL annealed linkers (made of the annealing of the following oligos: oligo 1, GCGGTGACCCGGGAGATCTGAATTC and oligo 2, GAATTCAGATC) and 0.5 µL (2.5 units) of T4 DNA ligase (ThermoFisher, 15224041), overnight at 16˚C. Samples were precipitated with 6 µL of 3M NaOAc pH 5.2 and ethanol, and resuspended in 25 µL H2O. 15 µL of labeling mix (4 µL 10X ThermoPol buffer, 2 µL of 5 mM a.a.-dUTP/dNTP mix (5mM each dATP, dGTP and dCTP, 3mM dTTP and 2mM aminoallyl-dUTP) and 1.25 µL of oligo 1 (from 40 µM stock) was add to each sample. Samples were placed in a thermocycler and temperature was raised to 55˚C.  10 µL of enzyme mix (8 µL H2O, 1 µL 10X ThermoPoll buffer, 1 µL (5 units) Taq polymerase (ThermoFisher, 18038240) and 0.01 µL (0.025 units) Pfu (Fermentas, EP0502)) was added and the following program was run. 1) 72˚C, 5 min, 2) 95˚C, 2 min, 3) 31 cycles: 95˚C, 30sec; 55˚C, 30sec; 72˚C, 1min, 4) 72˚C, 4min, 5) 4˚C, forever. PCR products were purified using QIAgen PCR purification kit with few modifications: PE buffer was replaced by a Phosphate wash buffer (5 mM KPO4 pH 8.5 and 80% ethanol) and EB buffer was replace by Phosphate elution buffer (4 mM KPO4 pH 8.5). Supernatants were dried in a speed-vac and pellets were resuspended in 4.5 µL of freshly prepared 0.1 M Na2CO3 pH 9.0 buffer. 4.5 µL of Cy5 (ChIP samples) or Cy3 (control samples)-ester dye (GE Healthcare, CA95017-373L and CA95017-379L) were added. Samples were incubated 1 hr at room temperature. 35 µL of 0.1 M NaOAc pH 5.2 was added and samples were purified on QIAgen PCR purification kit (28106) using the manufacturer’s protocol. ChIP and control samples were combined and dried in a speed-vac.

growth_protocol:
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.
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.

extract_protocol: Frozen cell pellets were resuspended in 700 µL of Lysis buffer (50 mM HEPES-KOH 1 M pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % Na-deoxycholate) supplemented with protease inhibitors (1 mM PMSF, 1 mM benzamidine, 10 µg/mL aprotinin, 1 µg/mL leupeptin and 1 µg/mL pepstatin) and lysed by bead beating. The extracts were sonicated with a Model 100 Sonic dismembrator equipped with a micro probe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles to obtain fragments averaging 500 bp. Samples were centrifuged at 20,000 g for 5 min at 4˚C to remove cell debris and 500 µL of soluble chromatin were used per ChIP experiment. 5 µL (1% of the amount used for IP) was also kept as an Input sample.

tag: strain

tag: genotype

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR508 (CEN, HIS3, rpb1-CTDT4V-3xFLAG)]

tag: antibody

value: 9E10 (lab stock by AF)


title: CTDY1F-rep2

description:
Biological replicate 2 of 2. Rpb1-CTDY1F-3xFlag occupancy using NET-seq in Rpb1-CTDWT-3xMyc and Rpb1-CTDY1F-3xFlag cells. Rpb1-CTDY1F-3xFlag NET-seq sample was performed using a mouse monoclonal antibody against Flag.
processed data file: Y1F-2_R1-negative.bigwig
processed data file: Y1F-2_R1-positive.bigwig
processed data file: Y1F-negative.bigwig
processed data file: Y1F-positive.bigwig

library_source: transcriptomic

data_processing:
Reads were aligned to the S. cerevisiae (sacCer3) genome using TopHat2 (Kim et al., 2013) with the following parameters: Read mismatches = 3, Read gap length = 2, Read edit distance = 3, Minimum anchor length = 8, Splice mismatches = 1, Minimum intron length = 50, Maximum intron length = 1200, Maximum insertion length = 3, Maximum deletion length = 3, Maximum multihits = 100, Segment mismatches = 3, Minimum coverage intron = 50, Maximum coverage intron = 100000, Minimum segment intron = 50, Maximum segment intron = 500000. The resulting BAM files were converted to BED files using Bamtools (Barnett et al., 2011) and then to bigBed using ucsc_tools (http://hgdownload.soe.ucsc.edu/downloads.html#source_downloads). The reads were trimmed to keep only the first sequenced base using an in-house tool available at https://github.com/IRCM/bed-tools-j/releases/tag/1. Coverage for each base pair of the S. cerevisiae genome (on each strand) was computed using genomeCoverageBed from BEDTools (version 2.19.1) (Quinlan and Hall, 2010) and normalized to 100 million uniquely mapped reads (as determined by running Flagstat from Samtools (version 1.2) (Li et al., 2009) on the BAM files). BedGraphs were then converted to bigWig (using Wig/BedGraph-to-bigWig from ucsc_tools) for data visualization on the UCSC Genome Browser (Casper et al., 2018) and for downstream analyses (see below). Replicate samples showed high correlation and were combined by merging BAM files using MergeSamFiles from Picard Tools (version 1.56.0) (http://broadinstitute.github.io/picard/) prior to calculation of the coverage and normalization.
Genome_build: Saccharomyces cerevisiae (sacCer3)
Supplementary_files_format_and_content: Normalized BigWig files (ending in “_R1-positive.bigwig” or  “_R1-negative.bigwig”) represent individual replicates.  Combined normalized BigWig files (ending in “-positive.bigwig” or “-negative.bigwig”) represent combined replicates.

source_name: Frozen yeast cells

growth_protocol: Yeast cells were inoculated at OD600=0.1 in 1 L of YNB –HIS supplemented with 2% glucose and grown at 30˚C. When cells reached OD600 0.7-0.8, they were harvested by microfiltration. Cells were scraped off the filter and transferred into a 50 mL canonical tube containing liquid nitrogen. Liquid nitrogen was removed and cells were stored at -80˚C.

extract_protocol:
Frozen cells were cryogenically lysed in a mixer mill (Retasch PM100) using the following parameters: 8 cycles at 500 rpm during 3 min with 1 min reverse rotation without breaks between rotations, keeping cells frozen at all time. Frozen cell grindates were stored at -80˚C. 1 g of cell grindate was resuspended in 5 mL of ice-cold Lysis buffer (20 mM HEPES pH 7.4, 110 mM KOAc, 0.5% Triton X-100, 0.1% Tween-20, 10 nM MnCl2, 1 mM PMSF, 1 mM benzamidine, 10 µg/mL aprotinin, 1 µg/mL leupeptin and 1 µg/mL pepstatin) by pipetting. 660 units of RNase-free DNase I (Promega, MG101) were added to the mix and the sample was incubated 20 min on ice. The lysate was then centrifuged for 10 min at 20,000 g at 4˚C. 300 µL of Dynabeads coated with Pan Mouse IgG antibodies (Thermo Fisher Scientific, 11042), pre-coupled to 30 µg of anti-Flag antibody (Sigma-Aldrich, F3165) were added to the supernatant which was incubated 2.5 hr at 4˚C on rotator.  Beads were washed four times with 10 mL of ice-cold Wash buffer (20 mM HEPES pH 7.4, 110 mM KOAc, 0.5% Triton X-100, 0.1% Tween-20, 1 mM EDTA). After the last wash, beads were transferred into a 1.5 mL microcentrifuge tube and placed on a magnet to discard any remaining supernatant. To eluate the IP proteins, 120 mg of 3xFlag peptide (Sigma-Aldrich, F4799-4MG) in 600 µL of ice-cold Lysis buffer was added to the beads which were incubated 30 min on ice. A second elution was performed and both supernatants were combined. The immunoprecipitated RNA was purified using the QIAgen miRNeasy mini kit (217004) according to manufacturer’s protocol with the following modifications: 1.5 volumes of QIAzol lysis reagent were used and two elutions with 50 µL nuclease-free water were done and pooled. RNA was quantified on a Nanodrop (ThermoFisher), precipitated with ethanol and resuspended in 11 µL of 10 mM Tris-HCl pH 7.0.
To construct a DNA sequencing library from the 3’ ends of a nascent RNA sample, 5 µL of RNA (1.5 µg) in 10 mM Tris-HCl pH 7.0 was denatured for 2 min at 80˚C in a thermocycler and placed on ice. DNA adapters were ligated to each RNA sample by adding 14 µL ligation reaction mix (8 µL 50% PEG M.W. 8,000, 2 µL 10X RNL2 buffer, 1 µL DNA linker (0.5 µg/µL), 3 µL nuclease-free H2O and 1 µL (200 units) T4 RNA ligase 2 (truncated, K227Q; New England Biolabs M0351S) and incubated overnight at 16˚C. Tubes were placed on ice and 0.7 µL 0.5 M EDTA was added. Then, 20 µL of 2X alkaline fragmentation solution (100 mM NaCO3 pH 9.2, 2 mM EDTA) was added to the ligated RNA samples which were incubated at 95˚C for a pre-determined calibrated time (0-50 min). The fragmented RNA was precipitated with isopropanol, washed with 70% ethanol and resuspended in 10 µL 10 mM Tris-HCl  pH 7.0. The RNA was run on 15% TBE-urea polyacrylamide gel, stained with SYBR gold. Dark Reader DR-45M2 transilluminator (Clare Chemical Research) was used to visualize the RNA and the gel piece corresponding to the ligated oligonucleotide (between 45 nt and 95 nt) was excised. Gel pieces were placed into 0.5 mL RNase-free, non-stick microcentrifuge tubes that had been needle-pierced and placed inside 1.5 mL RNase-free non-stick tubes.  Tubes were centrifuged 3 min at 20,000 g at room temperature to force the gel through the needle hole. 200 µL RNase-free H2O was added, followed by 10 min incubation at 70˚C. The gel slurry was vortexed for 30 sec, transferred to a Spin-X® centrifuge tube filter (Costar) and centrifuged 3 min at 20,000 g at room temperature to recover the elution mixture. RNA was ethanol-precipitated and resuspended in 10 µL of 10 mM Tris-HCl pH 7.0. RNA samples were then reverse transcribed as follow. 4.6 µL of RT reaction mix (3.28 µL 5X first strand buffer, 0.82 µL dNTPs (10 mM) and 0.5 µL RT primer) was added to samples, which were denatured 2 min at 80˚C and placed on ice. A mix of 0.5 µL  (0.01 units) SUPERase In (ThermoFisher AM2694) and 0.82 µL 0.1 M DTT was added together with 0.82 µL (170 units) Superscript IV (ThermoFisher 18090050) and samples were incubated 30 min at 48˚C. To degrade RNA, 1.8 µL 1 M NaOH was added and samples were incubated 20 min at 98˚C. Reactions were neutralized by adding 1.8 µL 1 M HCl. Samples were run on 10% TBE-urea polyacrylamide gel, bands were excised and DNA was extracted as described above. cDNA samples were precipitated with isopropanol and resuspended in 15 µL of 10 mM Tris-HCl pH 7.5. A mix of (2 µL 10X circ buffer, 1 µL 1 mM ATP, 1 µL 50 mM MnCl2 and 1 µL (100 units) CircLigase (Mandel LGN-CL4111K)) was added to cDNA and samples were incubated 1 hr at 60˚C to circularize DNA. Samples were incubated 10 min at 80˚C to heat-inactivate the enzyme. 5 µL of circularized DNA was added to 79.2 µL of PCR master mix (16.7 µL 5X Phusion HF buffer, 0.7 µL 10 mM dNTPs, 0.8 µL 50 µM reverse PCR primer, 0.4 µL 100 µM forward primer, 58.8 µL RNase-free H2O and 0.8 µL (1.6 units) Phusion polymerase (New England Biolabs M0530S)) and PCR was performed as follows: 1) 98˚C, 30 sec; 2) 6, 8, 10 and 12 cycles of 98˚C, 10 sec/60˚C, 10 sec/ 72˚C, 5 sec; 3) 4˚C, forever. PCR products were run on 8% TBE gel and stained with SYBR gold. The appropriate number of cycles (6, 8, 10 or 12 cycles) was determined and the PCR was repeated on the remaining of the sample using the selected number of cycle. PCR bands were cut and centrifuged through a needle hole as described above. Crunched gels were soaked in 670 µL DNA soaking buffer (0.3 M NaCl, 10 mM Tris-HCl pH 8.0 and 0.97 mM EDTA) overnight with agitation at room temperature. The gel mixture was transferred to a Spin-X® centrifuge tube filter (Costar) and centrifuged 30 min at 20,000 g at room temperature. Samples were precipitated with isopropanol and resuspended in 7 µL of 10 mM Tris-HCl pH 8.0. Library concentration and size (expected to be in the 175-225 bp range) were determined on a 2100 Bioanalyzer (Agilent). Sequencing was performed on an Illumina HiSeq 2500 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp singled-end reads.
NET-seq

tag: strain name

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR511 (CEN, HIS3, rpb1-CTDY1F-3xFLAG)]

value: Flag M2 "R" (Sigma)


title: CTDY1F-rep1

description:
Biological replicate 1 of 2. Rpb1-CTDY1F-3xFlag occupancy using NET-seq in Rpb1-CTDWT-3xMyc and Rpb1-CTDY1F-3xFlag cells. Rpb1-CTDY1F-3xFlag NET-seq sample was performed using a mouse monoclonal antibody against Flag.
processed data file: Y1F-1_R1-negative.bigwig
processed data file: Y1F-1_R1-positive.bigwig
processed data file: Y1F-negative.bigwig
processed data file: Y1F-positive.bigwig


title: yFR2223_CTDWT-3xWT-endo_MYCvsInput_20151125_rep2

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. rpb1-CTDY1D-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells. CTDY1D ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2223_CTDY1D-3xFLAG-Exo_FLAGvsInput_20151125_rep1-2__S.cerevisiaeApr11_72130.bed
A genome browser-ready file for CTDY1D occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR574 (CEN, HIS3, rpb1-CTDY1D-3xFLAG)]


title: yFR1669_MYCvsInput_CTDwt-Endo-rep1

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1669_MYCvsInput_CTDwt-Endo-rep1-2_20140220__S.cerevisiaeApr11_448927.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1630_MYCvsInput_CTDwt-Endo-rep1_20140114

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1630_MYCvsInput_CTDwt-Endo-rep1-3_20140310__S.cerevisiaeApr11_364206.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1630_FLAGvsInput_CTDY1F-Exo-rep1_20140114

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 1 of 2. rpb1-CTDY1F-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells. CTDY1F ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1630_FLAGvsInput_CTDY1F-Exo-rep1-2_20140130__S.cerevisiaeApr11_8261.bed
A genome browser-ready file for CTDY1F occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: Flag M2 R (Sigma)


title: yFR2223_CTDWT-3xWT-endo_MYCvsInput_20151125_rep1

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 1 of 2. rpb1-CTDY1D-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells. CTDY1D ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2223_CTDY1D-3xFLAG-Exo_FLAGvsInput_20151125_rep1-2__S.cerevisiaeApr11_72130.bed
A genome browser-ready file for CTDY1D occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR2223_CTDY1D-3xFLAG-Exo_FLAGvsInput_20151125_rep1

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2223_CTDWT-3xWT-endo_MYCvsInput_20151125_rep1-2__S.cerevisiaeApr11_238286.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR2223_CTDY1D-3xFLAG-Exo_FLAGvsInput_20151125_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2223_CTDWT-3xWT-endo_MYCvsInput_20151125_rep1-2__S.cerevisiaeApr11_238286.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1D-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: CTDWT-rep2

description:
Biological replicate 2 of 2. Rpb1-CTDWT-3xFlag occupancy using NET-seq in Rpb1-CTDWT-3xMyc and Rpb1-CTDWT-3xFlag cells. Rpb1-CTDWT-3xFlag NET-seq sample was performed using a mouse monoclonal antibody against Flag.
processed data file: WT-2_R1-negative.bigwig
processed data file: WT-2_R1-positive.bigwig
processed data file: WT-negative.bigwig
processed data file: WT-positive.bigwig

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR482 (CEN, HIS3, RPB1-CTDWT-3xFLAG)]


title: yFR2227_CTDWT-3xWT-endo_MYCvsInput_20151125_rep1

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 1 of 2. rpb1-CTDY1E-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1E-3xFLAG cells. CTDY1E ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2227_CTDY1E-3xFLAG-Exo_FLAGvsInput_20151125_rep1-2__S.cerevisiaeApr11_475617.bed
A genome browser-ready file for CTDY1E occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1E-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR576 (CEN, HIS3, rpb1-CTDY1E-3xFLAG)]


title: yFR1669_FLAGvsInput_CTDT4V-Exo-rep2

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. rpb1-CTDT4V-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells. CTDT4V ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1669_FLAGvsInput_CTDT4V-Exo-rep1-2_20140220__S.cerevisiaeApr11_560969.bed
A genome browser-ready file for CTDT4V occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1630_FLAGvsInput_CTDY1F-Exo-rep2_20140114

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. rpb1-CTDY1F-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells. CTDY1F ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1630_FLAGvsInput_CTDY1F-Exo-rep1-2_20140130__S.cerevisiaeApr11_8261.bed
A genome browser-ready file for CTDY1F occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1523_FLAGvsInput_CTDS7A-Exo-rep2_20140130

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. rpb1-CTDS7A-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS7A-3xFLAG cells. CTDS7A ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1523_FLAGvsInput_CTDS7A-Exo-rep1-2_20140130__S.cerevisiaeApr11_954346.bed
A genome browser-ready file for CTDS7A occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS7A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR494 (CEN, HIS3, rpb1-CTDS7A-3xFLAG)]


title: yFR2227_CTDWT-3xWT-endo_MYCvsInput_20151125_rep2

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. rpb1-CTDY1E-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1E-3xFLAG cells. CTDY1E ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR2227_CTDY1E-3xFLAG-Exo_FLAGvsInput_20151125_rep1-2__S.cerevisiaeApr11_475617.bed
A genome browser-ready file for CTDY1E occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1E-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1510_FLAGvsInput_CTD-S5A-Exo_rep2

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. rpb1-CTDS5A-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells. CTDS5A ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1510_FLAGvsInput_CTD-S5A-Exo_rep1-2__S.cerevisiaeApr11_949479.bed
A genome browser-ready file for CTDS5A occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR490 (CEN, HIS3, rpb1-CTDS5A-3xFLAG)]


title: yFR1523_MYCvsInput_CTDwt-Endo-rep2_20140130

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS7A-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1523_MYCvsInput_CTDwt-Endo-rep1-2_20140130__S.cerevisiaeApr11_314755.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS7A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: CTDWT-rep1

description:
Biological replicate 1 of 2. Rpb1-CTDWT-3xFlag occupancy using NET-seq in Rpb1-CTDWT-3xMyc and Rpb1-CTDWT-3xFlag cells. Rpb1-CTDWT-3xFlag NET-seq sample was performed using a mouse monoclonal antibody against Flag.
processed data file: WT-1_R1-negative.bigwig
processed data file: WT-1_R1-positive.bigwig
processed data file: WT-negative.bigwig
processed data file: WT-positive.bigwig


title: yFR1510_MYCvsInput_CTDwt-Endo_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1510_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_750184.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1508_MYCvsInput_CTDwt-Endo_rep1

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS2A-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1508_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_823628.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS2A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR486 (CEN, HIS3, rpb1-CTDS2A-3xFLAG)]


title: yFR1630_MYCvsInput_CTDwt-Endo-rep2_20140114

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1630_MYCvsInput_CTDwt-Endo-rep1-3_20140310__S.cerevisiaeApr11_364206.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDY1F-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1523_MYCvsInput_CTDwt-Endo-rep1_20140130

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS7A-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1523_MYCvsInput_CTDwt-Endo-rep1-2_20140130__S.cerevisiaeApr11_314755.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS7A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1669_FLAGvsInput_CTDT4V-Exo-rep1

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 1 of 2. rpb1-CTDT4V-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells. CTDT4V ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1669_FLAGvsInput_CTDT4V-Exo-rep1-2_20140220__S.cerevisiaeApr11_560969.bed
A genome browser-ready file for CTDT4V occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDT4V-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1510_FLAGvsInput_CTD-S5A-Exo_rep1

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 1 of 2. rpb1-CTDS5A-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells. CTDS5A ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1510_FLAGvsInput_CTD-S5A-Exo_rep1-2__S.cerevisiaeApr11_949479.bed
A genome browser-ready file for CTDS5A occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1510_MYCvsInput_CTDwt-Endo_rep1

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1510_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_750184.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS5A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1508_MYCvsInput_CTDwt-Endo_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS2A-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1508_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_823628.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS2A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1508_FLAGvsInput_CTD-S2A-Exo_rep2

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. rpb1-CTDS2A-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDS2A-3xFLAG cells. CTDS2A ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1508_FLAGvsInput_CTD-S2A-Exo_rep1-2__S.cerevisiaeApr11_468185.bed
A genome browser-ready file for CTDS2A occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDS2A-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1506_MYCvsInput_CTDwt-Endo_rep2

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 2 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-Rpb1-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1506_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_729997.bed
A genome browser-ready file for endo-CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and endo-RPB1-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: yFR1506_FLAGvsInput_CTDwt-Exo_rep2

description:
Anti-Flag M2 (Sigma F3165). Anti-mouse Dynabeads (PanMouse) (50uL / ChIP) are washed twice with 10mL PBS + 0.5% BSA (freshly made), and resuspended in 1mL 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.
Biological replicate 2 of 2. Rpb1-3xFLAG occupancy in endo-Rpb1-3xMYC and exo-Rpb1-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against FLAG and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1506_FLAGvsInput_CTDwt-Exo_rep1-2__S.cerevisiaeApr11_95606.bed
A genome browser-ready file for exo-CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and endo-RPB1-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.


title: SC2_2_AB1175

city: Bethesda

first: Timothy

middle: G

last: Myers

state: MD

address: 50 South Drive, Room 5509

department: Research Technologies Branch

institute: National Institute of Allergy and Infectious Diseases

description: Lentivirus KD scrambled control 2

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol with Illumina HiScan-SQ

data_processing: Signal data was extracted from the image files with the Gene Expression module (v. 1.9.0) of the GenomeStudio software (v. 2011.1) from Illumina, Inc. Signal intensities were converted to log 2 scale. Probes that did not have detection p-value < 0.1 in at least 2 arrays were removed from the dataset.  After filtering probes, quantile normalization was applied across all arrays.

organism: Homo sapiens

source_name: HEK239-Kb cells

label_protocol: Illumina TotalPrep RNA Amplification Kit (Ambion)

growth_protocol: Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS)

extract_protocol: RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit.

treatment_protocol: Transduce 293Kb cells (HEK293 cells expressing the mouse class I molecule H-2Kb from a transgene) with lentiviruses and total RNA extracted 6 days after lentivirus transduction

tag: exp


title: yFR1376_MYCvsInput_CTDwt-Endo_rep1

description:
9E10 anti-Myc antibody. Anti-mouse Dynabeads (PanMouse) (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 PBS/BSA or Lysis buffer), 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. 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.
Biological replicate 1 of 2. Rpb1-3xMYC occupancy in endo-Rpb1-3xMYC and exo-rpb1-CTDless-3xFLAG cells. CTDWT ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against MYC and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR1376_MYCvsInput_CTDwt-Endo_rep1-2__S.cerevisiaeApr11_733790.bed
A genome browser-ready file for CTDWT occupancy (IP/Input) in endo-RPB1-3xMYC and exo-rpb1-CTDless-3xFLAG cells.
The ChIP-chip data from individual replicates were normalized by setting the median of log2 ratios to zero. The duplicate were averaged and the data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100bp to generate data points in 10bp intervals as described before (Guillemette et al., 2005, PLoS Biol 3(12):e384).
Supernatants were recovered and treated with RNase A (345 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA), 3 μL RNAse A 10 mg/mL, 2 μL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. DNA was extracted twice with phenol/chloroform/isoamyl alcool, ethanol-precipitated and resuspended in 50 µL TE.

value: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL+, psi+, RPB1-3xMYC::URA3, [pFR467 (CEN, HIS3, rpb1-CTDless-3xFLAG)]


title: SC1_2_AB1173

description: Lentivirus KD scrambled control 1


title: RPL6_2_AB1165

description: Lentivirus KD targeting RPL6


title: RPL28_2_AB1167

description: Lentivirus KD targeting RPL28