title: ChIPexo_IgG_chd1D_R1

description: Biological replicate 1 of 1. IgG occupancy by ChIP-exo in chd1D cells. IgG ChIP-exo was performed using normal rabbit IgG polyclonal antibody control.

data_processing:
Paired-end reads from ChIP-exo data were aligned to the yeast S. cerevisiae genome (sacCer3 from UCSC Genome Browser) using the BWA version 0.7.17 (Li, 2013). Unmapped reads, reads not mapped properly in pairs, and duplicates were removed from the analysis using SAMtools version 1.8 (Li et al., 2009). For each paired-end read, only the first read was kept for further analysis. The aligned files were converted to BED using BEDTools version 2.28.0 (Quinlan and Hall, 2010). BED files containing reads from replicates were further analyzed individually in addition to being merged. The reads were moved 6 bp towards the 3’ end to correct for the offset of exonuclease stops and the site of crosslinking using a custom java script called bed-tools-j, version 2.1 – https://github.com/francoisrobertlab/bed-tools-j/releases/tag/2.1. Three genome coverage files were computed using the 5’ position of the moved reads using BEDTools. Coverage files were generated for reads mapping the positive strand, the negative strand, and the merged strands. The genome coverage files were converted to the bigWig format using the utilities from UCSC Genome Browser (Casper et al., 2018). The analysis was partially automated using a custom script called seqtools available at https://github.com/francoisrobertlab/seqtools.
For each biological replicate, three genome coverage files (bigWig) for reads mapping the positive strand (ending with "pos.bw"), the negative strand (ending with "neg.bw"), and the merged strands (ending with ".bw"). In addition, a coverage file (merge of both strands) is provided for the combined biological replicates (beginning with "ChIPexo").
Genome_build: S. cerevisiae (UCSC sacCer3)
Supplementary_files_format_and_content: bigWig

growth_protocol: Cells were grown at 30°C and 200 rpm in YPD (yeast extract-peptone-2% glucose, supplemented with 44 µM adenine) medium as follow. Strains were streaked from glycerol stocks onto 2% agar YPD plates and grown at 30°C for 2-3 days. An isolated colony was then grown overnight in 10 mL of YPD. This preculture was used to inoculate 50 mL of YPD at an OD600 of 0.1 which was grown to an OD600 of 0.7-0.9.

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D704)
Barcode ID #2:  2nd  ligation adapter (D503)
ChIP-exo

value: rabbit IgG (Millipore 12-370, 2.5 μg)


title: cell36

status: Public on Jul 26 2021

type: SRA

city: Beijing

first: Zeyuan

last: Liu

address: 15 Datun Road, Chaoyang District, Beijing, 100101, China

country: China

institute: Institute of Biophysics,Chinese Academy of Sciences

description:
rep7-InG_ZI27
smart-seq-raw-counts-for-geo.txt

library_source: transcriptomic

data_processing:
Illumina Casava1.8 software used for basecalling
Reads were aligned to GRCm38 mouse genome and mm10 mouse genome by STAR(v2.5.3a) and Cellranger(v3.0.2)
Filtered count matrix was merged by Seurat Package
Genome_build: GRCm38, mm10
Supplementary_files_format_and_content: Filtered count matrix was formatted by mtx and txt file, and barcode and gene information was with tsv format

molecule: total RNA

organism: Mus musculus

source_name: mouse brain

extract_protocol:
Mouse brain samples were collected in ice-cold artificial cerebrospinal fluid (ACSF), single nuclei were extracted by lysis buffer
Chromium™ Single Cell 3’ Reagent Kits v3

tag: age

value: 3 months

tag: tissue

value: brain

tag: region

value: superior colliculus

tag: sequencing stragery


title: NDB06_b2_6mo_G7A01_b1_6mo

city: New York

first: Alison

last: Goate

state: NY

address: Icahn (East) Building Floor 10 Room 10-70C, 1425 Madison Ave

department: Department of Genetics and Genomic Sciences

country: USA

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Digital expression was carried out using Drop-Seq tools
STAR v2.5.2a was used for read alignments
HTO antibodies were counted using CITE-Seq Count (v1.4.2) and demultiplexed using Seurat v3.0
Cells with <200 detectable genes, <500 reads and >20% mitochondrial rate were removed using Seurat v3.0(dev)
Data were integrated using SCTransform (Seurat v3.0 (dev)) and annotated using SingleR with custom reference libraries built from Polioudakis et al. Neuron, 2019 and Zhang et al. Neuron, 2016
Genome_build: Hg19
Supplementary_files_format_and_content: Gene read counts per cell

molecule: polyA RNA

organism: Homo sapiens

source_name: iPSC-derived cerebral organoid

growth_protocol: Cerebral organoids were generated as per previously published protocol - see Yoon et al. Nature Methods, 2019.

extract_protocol:
Organoids were dissociated into single cell suspensions using Papain (Worthington). Resulting cells were labelled with hash-tag oligonucleotide antibodies as described in Stoeckius et al. Genome Biology, 2018.
Libraries were prepared and sequenced using either the 10x Genomics Single Cell v2 or v3 platforms. Libraries were sequenced with either a 26x90bp or 28x90bp read length
Single-cell RNA sequencing

treatment_protocol: No treatment

tag: cell line

value: 6 months

tag: batch

tag: genotype


title: cell34

description:
rep5-InG_ZI21
smart-seq-raw-counts-for-geo.txt


title: ChIPexo_Pob3_chd1D_R2

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

country: Canada

institute: IRCM

description:
Biological replicate 2 of 2. Pob3 occupancy by ChIP-exo in WT cells. Pob3 ChIP-exo was performed using a rabbit polyclonal antibody against Pob3.
processed data file (combined biological replicates):  ChIPexo_Pob3_yFR930chd1D_CJ11-CJ15_20190412.bw

library_source: genomic

molecule: genomic DNA

organism: Saccharomyces cerevisiae

source_name: Yeast cell extract

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D703)
Barcode ID #2:  2nd  ligation adapter (D504)
ChIP-exo

treatment_protocol: Cultures were crosslinked with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min. Crosslinking was quenched with 125 mM glycine.

tag: strain name

tag: chip antibody

value: anti-Pob3 (a gift from T. Formosa, 1 μL)


title: G7B12_b1_6mo_G7F02_b1_6mo


title: yFR116_WT_Rpb3vsInput_repl2

type: genomic

description:
Biological replicate 2 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_Rep1-2_S.cerevisiaeApr11_271024.bed

hyb_protocol: ChIP and control samples were combined and dried by SpeedVac. Combined samples were resuspended in 110 µL of fresh hybridization mix (5 µL 10 mg/mL Salmon Sperm DNA (Thermo Fisher Scientific, 15632011), 5 µL 8 mg/mL Yeast tRNA (Thermo Fisher Scientific, 15401011) and 100 µL DIG Easy Hyb (Roche, 11603558001)) and incubated 5 min at 95˚C followed by 5 min at 45˚C. Samples were hybridized to Agilent microarrays in SureHyb enabled chambers (Agilent, G2534A) with rotation at 42˚C for at least 18 hr. Microarrays were washed on an orbital shaker for 5 min at room temperature with Wash I buffer (6X SSPE, 0.005% N-Lauroylsarcosine), followed by a second wash with pre-warmed (31˚C) Wash II buffer (0.06X SSPE) for 5 min at room temperature. The microarrays used were described before (Jeronimo and Robert, 2014) and were custom designed by Agilent Technologies and contain a total of about 180,000 Tm-adjusted 60-mer probes covering the entire yeast genome with virtually no gaps between probes.

scan_protocol: Microarrays were scanned using an InnoScan900 (Innopsys) at 2 µm resolution.

data_processing:
BED description:  A genome browser-ready file for Rpb3 occupancy by ChIP-chip (IP/Input) in WT cells.
BED method:  The ChIP-chip data were normalized using the Limma Loess method, and replicates were combined as described previously (Ren et al. 2000, Science 290(5500):2306-9). The data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100 bp to generate data points in 10-bp intervals (Guillemette et al. 2005, PLoS Biol 3(12):e384).
Correction = Foreground-Background; Normalization = limma loess (subgrid)

label_protocol: The ChIP and Input samples were blunted as follow. 40 µL of ChIP DNA or about 400 ng of Input DNA were added to 70 µL of blunting¬¬ mix (11 µL 10X NEBuffer 2 (NEB, B7002S), 0.5 µL 10 mg/mL BSA, 0.5 µL 20 mM dNTPs and 0.2 µL (0.6 units) of T4 DNA polymerase (NEB, M0203L)) and incubated at 12˚C for 20 min. Then, the blunted DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) in the presence of 280 mM of NaOAc pH 5.2 and 10 μg of glycogen, precipitated with ethanol and resuspended in 25 µL ddH2O. Blunted DNA was ligated to 100 pmol of annealed linkers (see Table S2) with 2.5 units of T4 DNA ligase (Thermo Fisher Scientific, 15224041) in the supplied buffer, in a final volume of 50 µL, overnight at 16˚C. Samples were precipitated with 6 µL of 3 M NaOAc pH 5.2 and 130 µL ethanol, and resuspended in 25 µL ddH2O. 15 µL of labeling mix (4 µL 10X ThermoPol Reaction buffer (NEB, B9004S), 2 µL of 5 mM aa-dUTP/dNTP mix (5 mM each dATP, dGTP and dCTP, 3 mM dTTP and 2 mM 5-(3-aminoallyl)-dUTP (Thermo Fisher Scientific, AM8439)) and 1.25 µL of 40 µM oligo 1) were added to each sample. Samples were placed in a thermocycler and when temperature reached 55˚C, 10 µL of enzyme mix (1 µL 10X ThermoPol Reaction buffer, 1 µL (5 units) Taq DNA polymerase (Thermo Fisher Scientific, 18038240) and 0.01 µL (0.025 units) Pfu DNA polymerase (Thermo Fisher Scientific, EP0502)) were added and the following program was run. 1) 72˚C, 5 min; 2) 95˚C, 2 min; 3) 32 cycles: 95˚C, 30sec; 55˚C, 30sec; 72˚C, 1 min; 4) 72˚C, 4 min; 5) 4˚C, forever. PCR products were purified using QIAquick PCR Purification kit (QIAGEN, 28106) with few modifications: PE buffer was replaced by 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). Eluates were dried by SpeedVac and pellets were resuspended in 4.5 µL of freshly prepared 0.1 M Na2CO3 pH 9.0 buffer. 4.5 µL of Cy5 Mono-Reactive NHS Ester Fluorescent Dye (ChIP samples) or Cy3 Mono-Reactive NHS Ester Fluorescent Dye (control samples Input DNA or no-tag ChIP) (GE Healthcare, PA25001 and PA23001) were added. Samples were incubated 1 hr in the dark at room temperature. 35 µL of 0.1 M NaOAc pH 5.2 were added and samples were purified using QIAquick PCR Purification kit according to the manufacturers’ instructions.

growth_protocol: 50 mL of YPD (yeast extract-peptone-2% glucose, supplemented with 44 µM adenine) medium was inoculated at OD600 0.1 from an overnight preculture in the same medium and allowed to grow to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min and quenched with 125 mM glycine. Crosslinked cells were collected by centrifugation and washed twice with 1X TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). Cell pellets were snap-freezed in liquid nitrogen and stored at -80°C.

extract_protocol:
IP protocol:  Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 600 μL of the chromatin sample was taken per immunoprecipitation (IP) and, when necessary, 6 μL (1%) were saved as Input sample. The following amount of antibody per IP was used: anti-Rpb3 (Neoclone W0012, 3 μL). 50 μL of Dynabeads coated with Pan Mouse IgG antibodies (Thermo Fisher Scientific, 11042) pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.
Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation.
Cleanup protocol:  Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.

tag: strain

value: anti-Rpb3 (Neoclone W0012, 3 uL)

extract_protocol:
IP protocol:  none
Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation.
Cleanup protocol:  Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.


title: yFR116_WT_Spt16vsInput_20150711_repl1

description:
Biological replicate 1 of 2. FACT (Spt16) occupancy by ChIP-chip in WT cells. Spt16 ChIP sample (Cy5-labeled) was performed using a rabbit polyclonal antibody against Spt16 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Spt16vsInput_20150711_rep1-2_S.cerevisiaeApr11_517749.bed

data_processing:
BED description:  A genome browser-ready file for Spt16 occupancy by ChIP-chip (IP/Input) in WT cells.
BED method:  The ChIP-chip data were normalized using the Limma Loess method, and replicates were combined as described previously (Ren et al. 2000, Science 290(5500):2306-9). The data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100 bp to generate data points in 10-bp intervals (Guillemette et al. 2005, PLoS Biol 3(12):e384).
Correction = Foreground-Background; Normalization = limma loess (subgrid)

extract_protocol:
IP protocol:  Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 600 μL of the chromatin sample was taken per immunoprecipitation (IP) and, when necessary, 6 μL (1%) were saved as Input sample. The following amount of antibody per IP was used: anti-Spt16 (a gift from T. Formosa, 1 μL). 50 μL of Dynabeads coated with Protein G (Thermo Fisher Scientific, 10004D) pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.
Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation.
Cleanup protocol:  Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.

value: anti-Spt16 (a gift from T. Formosa, 1 uL)


title: ChIPexo_Rpb3_chd1D_R2

description:
Biological replicate 2 of 2. RNAPII (Rpb3) occupancy by ChIP-exo in  chd1D cells. Rpb3 ChIP-exo was performed using a rabbit polyclonal antibody against Rpb3.
processed data file (combined biological replicates):  ChIPexo_Rpb3_yFR930chd1D_CJ10-CJ14_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D702)
Barcode ID #2:  2nd  ligation adapter (D504)
ChIP-exo

value: anti-Rpb3 (BioLegend 665004, 1.5 μL)


title: G6E11_b1_6mo_G6A02_b1_6mo


title: cell32

description:
rep5-OP_lp34
smart-seq-raw-counts-for-geo.txt


title: cell33

description:
rep5-InG_ZI20
smart-seq-raw-counts-for-geo.txt


title: NDB06_b2_6mo_NDB09_b2_6mo_G7A01_b1_6mo


title: ChIPexo_Pob3_chd1D_R1

description:
Biological replicate 1 of 2. Pob3 occupancy by ChIP-exo in  chd1D cells. Pob3 ChIP-exo was performed using a rabbit polyclonal antibody against Pob3.
processed data file (combined biological replicates):  ChIPexo_Pob3_yFR930chd1D_CJ11-CJ15_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D703)
Barcode ID #2:  2nd  ligation adapter (D503)
ChIP-exo


title: cell35

description:
rep7-InG_ZI25
smart-seq-raw-counts-for-geo.txt


title: cell39

description:
rep7-InG_ZI30
smart-seq-raw-counts-for-geo.txt


title: G7B12_b3_4mo_G7F02_b3_4mo

value: 4 months


title: ChIPexo_Rpb3_chd1D_R1

description:
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-exo in  chd1D cells. Rpb3 ChIP-exo was performed using a rabbit polyclonal antibody against Rpb3.
processed data file (combined biological replicates):  ChIPexo_Rpb3_yFR930chd1D_CJ10-CJ14_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D702)
Barcode ID #2:  2nd  ligation adapter (D503)
ChIP-exo


title: ChIPexo_IgG_WT_R1

description: Biological replicate 1 of 1. IgG occupancy by ChIP-exo in WT cells. IgG ChIP-exo was performed using normal rabbit IgG polyclonal antibody control.

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D704)
Barcode ID #2:  2nd  ligation adapter (D502)
ChIP-exo


title: cell30

description:
rep5-OP_lp30
smart-seq-raw-counts-for-geo.txt


title: ChIPexo_Spt16_chd1D_R2

description:
Biological replicate 2 of 2. Spt16 occupancy by ChIP-exo in  chd1D cells. Spt16 ChIP-exo was performed using a rabbit polyclonal antibody against Spt16.
processed data file (combined biological replicates):  ChIPexo_Spt16_yFR930chd1D_CJ9-CJ13_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D701)
Barcode ID #2:  2nd  ligation adapter (D504)
ChIP-exo

value: anti-Spt16 (a gift from T. Formosa, 1 μL)


title: G6E11_b3_4mo_G7A01_b3_4mo


title: cell29

description:
rep5-OP_lp29
smart-seq-raw-counts-for-geo.txt


title: G7A01_b2_4mo_G6A02_b3_4mo


title: G7F02_b2_4mo_G6A02_b2_4mo


title: yFR116_WT_Rpb3vsInput_20190211_repl2

description:
Biological replicate 2 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20190211_rep1-2_S.cerevisiaeApr11_136185.bed

extract_protocol:
IP protocol:  Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 600 μL of the chromatin sample was taken per immunoprecipitation (IP) and, when necessary, 6 μL (1%) were saved as Input sample. The following amount of antibody per IP was used: anti-Rpb3 (BioLegend 665004, 1.5 μL). 50 μL of Dynabeads coated with Pan Mouse IgG antibodies (Thermo Fisher Scientific, 11042) pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.
Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation.
Cleanup protocol:  Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.

value: anti-Rpb3 (BioLegend 665004, 1.5 uL)


title: yFR116_WT_Rpb3vsInput_repl1

description:
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_Rep1-2_S.cerevisiaeApr11_271024.bed


title: yFR116_WT_Rpb3vsInput_20190502_repl1

description:
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20190502_rep1-2_S.cerevisiaeApr11_888754.bed


title: yFR116_WT_Spt16vsInput_20150711_repl2

description:
Biological replicate 2 of 2. FACT (Spt16) occupancy by ChIP-chip in WT cells. Spt16 ChIP sample (Cy5-labeled) was performed using a rabbit polyclonal antibody against Spt16 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Spt16vsInput_20150711_rep1-2_S.cerevisiaeApr11_517749.bed


title: yFR116_WT_Rpb3vsInput_20190502_repl2

description:
Biological replicate 2 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20190502_rep1-2_S.cerevisiaeApr11_888754.bed


title: yFR116_WT_Rpb3vsInput_20160924_repl2

description:
Biological replicate 2 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20160924_rep1-2_S.cerevisiaeApr11_857054.bed


title: ChIPexo_Pob3_WT_R2

description:
Biological replicate 2 of 2. Pob3 occupancy by ChIP-exo in WT cells. Pob3 ChIP-exo was performed using a rabbit polyclonal antibody against Pob3.
processed data file (combined biological replicates):  ChIPexo_Pob3_yFR116WT_CJ3-CJ7_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D703)
Barcode ID #2:  2nd  ligation adapter (D502)
ChIP-exo


title: ChIPexo_Rpb3_WT_R2

description:
Biological replicate 2 of 2. RNAPII (Rpb3) occupancy by ChIP-exo in WT cells. Rpb3 ChIP-exo was performed using a rabbit polyclonal antibody against Rpb3.
processed data file (combined biological replicates):  ChIPexo_Rpb3_yFR116WT_CJ2-CJ6_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D702)
Barcode ID #2:  2nd  ligation adapter (D502)
ChIP-exo


title: yFR116_WT_Rpb3vsInput_20190211_repl1

description:
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20190211_rep1-2_S.cerevisiaeApr11_136185.bed


title: cell31

description:
rep5-OP_lp31
smart-seq-raw-counts-for-geo.txt


title: yFR116_WT_Rpb3vsInput_20160924_repl1

description:
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20160924_rep1-2_S.cerevisiaeApr11_857054.bed


title: cell28

description:
rep4-OP_lp27
smart-seq-raw-counts-for-geo.txt


title: ChIPexo_Spt16_WT_R2

description:
Biological replicate 2 of 2. Spt16 occupancy by ChIP-exo in WT cells. Spt16 ChIP-exo was performed using a rabbit polyclonal antibody against Spt16.
processed data file (combined biological replicates):  ChIPexo_Spt16_yFR116WT_CJ1-CJ5_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D701)
Barcode ID #2:  2nd  ligation adapter (D502)
ChIP-exo


title: cell26

description:
rep4-OP_lp23
smart-seq-raw-counts-for-geo.txt


title: cell27

description:
rep4-OP_lp24
smart-seq-raw-counts-for-geo.txt


title: ChIPexo_Pob3_WT_R1

description:
Biological replicate 1 of 2. Pob3 occupancy by ChIP-exo in WT cells. Pob3 ChIP-exo was performed using a rabbit polyclonal antibody against Pob3.
processed data file (combined biological replicates):  ChIPexo_Pob3_yFR116WT_CJ3-CJ7_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D703)
Barcode ID #2:  2nd  ligation adapter (D501)
ChIP-exo


title: G6E11_b2_4mo_G7B12_b2_4mo


title: cell25

description:
rep4-OP_lp22
smart-seq-raw-counts-for-geo.txt


title: G7A01_b1_4mo


title: ChIPexo_Spt16_chd1D_R1

description:
Biological replicate 1 of 2. Spt16 occupancy by ChIP-exo in chd1D cells. Spt16 ChIP-exo was performed using a rabbit polyclonal antibody against Spt16.
processed data file (combined biological replicates):  ChIPexo_Spt16_yFR930chd1D_CJ9-CJ13_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D701)
Barcode ID #2:  2nd  ligation adapter (D503)
ChIP-exo


title: NDB06_b3_4mo_NDB09_b3_4mo


title: NDB06_b2_4mo_NDB09_b2_4mo


title: yFR116_WT_Rpb3vsInput_20150711_repl1

description:
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20150711_rep1-2_S.cerevisiaeApr11_101027.bed


title: G7B12_b1_4mo_G7F02_b1_4mo


title: ChIPexo_Spt16_WT_R1

description:
Biological replicate 1 of 2. Spt16 occupancy by ChIP-exo in WT cells. Spt16 ChIP-exo was performed using a rabbit polyclonal antibody against Spt16.
processed data file (combined biological replicates):  ChIPexo_Spt16_yFR116WT_CJ1-CJ5_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D701)
Barcode ID #2:  2nd  ligation adapter (D501)
ChIP-exo


title: cell24

description:
rep3-OP_lp18
smart-seq-raw-counts-for-geo.txt

extract_protocol:
Mouse brain samples were collected in ice-cold artificial cerebrospinal fluid (ACSF), single nuclei were extracted by lysis buffer
Chromium™ Single Cell 3’ Reagent Kits v3


title: ChIPexo_Rpb3_WT_R1

description:
Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-exo in WT cells. Rpb3 ChIP-exo was performed using a rabbit polyclonal antibody against Rpb3.
processed data file (combined biological replicates):  ChIPexo_Rpb3_yFR116WT_CJ2-CJ6_20190412.bw

extract_protocol:
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1:  1st ligation adapter (D702)
Barcode ID #2:  2nd  ligation adapter (D501)
ChIP-exo


title: G6E11_b1_4mo_G6A02_b1_4mo

extract_protocol:
Organoids were dissociated into single cell suspensions using Papain (Worthington). Resulting cells were labelled with hash-tag oligonucleotide antibodies as described in Stoeckius et al. Genome Biology, 2018.
Libraries were prepared and sequenced using either the 10x Genomics Single Cell v2 or v3 platforms. Libraries were sequenced with either a 26x90bp or 28x90bp read length
Single-cell RNA sequencing


title: FT194_NMNAT-2_Polysome rep2.fastq.gz

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

description: Replicates merged

data_processing:
The RNA-seq reads were aligned to the hg38 human reference genome using the Bowtie software package (Langmead et al, 2009) .
Mapped reads were sorted and captured in bam files for further downstream analysis of differential gene expression and  bigwig files were generated to for presentation as browser tracks using custom R scripts.
Genome_build: hg38
Supplementary_files_format_and_content: BigWig

source_name: FT194 Cells

growth_protocol: OVCAR3 cells were cultured in RPMI media containing 10% FBS, 1% Pencillin-streptavidin, 1% Glutamax and 250 µg/mL G418 . FT194 cells were cutured in DMEM-F12 media containing 10% FBS and 150 µg/mL G418

extract_protocol:
Input RNA and polysome associated RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer’s instructions from 2 individual 15 cm plates per replicate per treatment.
The total RNA was then enriched for polyA+ RNA using Dynabeads Oligo(dT)25 (Invitrogen).  The polyA+ RNA was then used to generate RNA-seq libraries. The RNA-seq libraries were subjected to QC analyses (i.e., number of PCR cycles required to amplify each library, the final library yield, and the size distribution of final library DNA fragments) and sequenced using an Illumina Nextseq 500.

treatment_protocol: OVCAR3 cells were transfected with lentiviral particles encoding for GFP or NMNAT-2 and positive clones were selected using 500 µg/mL G418 The cells were maintained in 250  µg/mL G418 after selection. FT194 cells were transfected with lentiviral particles encoding for vector or NMNAT-2 and positive clones were selected and maintained in 250  µg/mL G418.

tag: genotype/variation

tag: rna isolated from

value: polysome fractions

tag: molecule subtype

value: cDNA from reverse transcribed RNA


title: yFR116_WT_Rpb3vsInput_20150711_repl2

description:
Biological replicate 2 of 2. RNAPII (Rpb3) occupancy by ChIP-chip in WT cells. Rpb3 ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against Rpb3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Rpb3vsInput_20150711_rep1-2_S.cerevisiaeApr11_101027.bed

extract_protocol:
IP protocol:  Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 600 μL of the chromatin sample was taken per immunoprecipitation (IP) and, when necessary, 6 μL (1%) were saved as Input sample. The following amount of antibody per IP was used: anti-Rpb3 (Neoclone W0012, 3 μL). 50 μL of Dynabeads coated with Pan Mouse IgG antibodies (Thermo Fisher Scientific, 11042) pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.
Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation.
Cleanup protocol:  Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.

extract_protocol:
IP protocol:  none
Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation.
Cleanup protocol:  Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.


title: FT194_NMNAT-2_Polysome rep1.fastq.gz

description: Replicates merged


title: yFR116_WT_Pob3vsInput_repl1

description:
Biological replicate 1 of 1. FACT (Pob3) occupancy by ChIP-chip in WT cells. Pob3 ChIP sample (Cy5-labeled) was performed using a rabbit polyclonal antibody against Pob3 and hybridized against Input DNA (Cy3-labeled).
SmthBED_yFR116_WT_Pob3vsInput_rep1_S.cerevisiaeApr11_818514.bed

data_processing:
BED description:  A genome browser-ready file for Pob3 occupancy by ChIP-chip (IP/Input) in WT cells.
BED method:  The ChIP-chip data were normalized using the Limma Loess method, and replicates were combined as described previously (Ren et al. 2000, Science 290(5500):2306-9). The data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100 bp to generate data points in 10-bp intervals (Guillemette et al. 2005, PLoS Biol 3(12):e384).
Correction = Foreground-Background; Normalization = limma loess (subgrid)

extract_protocol:
IP protocol:  Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 600 μL of the chromatin sample was taken per immunoprecipitation (IP) and, when necessary, 6 μL (1%) were saved as Input sample. The following amount of antibody per IP was used: anti-Pob3 (a gift from T. Formosa, 1 μL). 50 μL of Dynabeads coated with Protein G (Thermo Fisher Scientific, 10004D) pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.
Cell pellets were resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation.
Cleanup protocol:  Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.

value: anti-Pob3 (a gift from T. Formosa, 1 uL)


title: NDB06_b1_6mo_NDB09_b1_6mo