title: HER2 Cas9 sat A431 High Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO65


title: 50_oocyte_Intermediate-Hoechst

status: Public on Mar 23 2017

type: SRA

city: Basel

first: Ilya

middle: M.

last: Flyamer

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

library_source: genomic

data_processing:
Base calling was performed by VBCF NGS unit
Mapping: iterative mapping (see Imakaev et al, 2012) using bowtie2 with increased stringency parameters: --score-min -L 0.6,0.2--very-  sensitive
Read filtering: if two reads map to the same strands, and each side of the read is within 500 bp of any side of the other read, we retained only one copy of the read.
Additional filtering: we separated the genome into 500 bp bins. If any two bins were interacting more than once, only one interaction was counted. Since each region is only present in the single cell up to 4 times (in oocytes), we eliminated all 500 bp bins that form more than 8 unique interactions with other bins.
Filtered reads were then binned at different resolutions (we provide files at 200Kb resolution) using read start as a read position. No normalization was applied to the data.
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: List of all interactions at single contact, 40Kb or 200 Kb resolution

molecule: genomic DNA

organism: Mus musculus

source_name: oocyte

growth_protocol: Fully grown denuded GV oocytes were isolated from 2-3 month old C57BL/6J mice by puncturing of ovaries with hypodermic needles28. Oocytes were incubated for at least 2 hours before the procedure to ensure viability. Before formaldehyde fixation, the zona pellucida was removed by treatment with Acidic Tyrode's solution (Sigma).

extract_protocol:
Oocytes were fixed in with 2% formaldehyde for 15 minutes at room temperature. Oocytes were washed through drops of complete M2. For SN and NSN distinction oocytes were stained for 15 min with 0.2 µg/ml Hoechst (33342, Invitrogen). Cells were then imaged using a LSM510 (Zeiss) confocal microscope and sorted according to their chromatin structure before proceeding with the Hi-C protocol. After fixation, single oocytes were incubated in 9 μl of lysis buffer for at least 15 minutes on ice. Similarly cells were incubated in 9 μl of 1× NEB3 buffer supplemented with 0.6% SDS for 2 hours at 37° with shaking in humidified atmosphere. Then cells were incubated in 1× DpnII buffer (NEB) with 1× BSA and  5 U DpnII (NEB). After overnight incubation in a humidified incubator at 37° the DNA ends were ligated in 9 μl of 1× T4 DNA ligase buffer containing 5 U T4 DNA ligase (Thermo Scientific) and they were incubated at 16°C for 4.5 hours with 50 rpm rotation, and then were kept for 30 min at room temperature. DNA from each nucleus was amplified using illustra GenomiPhi v2 DNA amplification kit (GE Healthcare) or illustra Single Cell GenomiPhi DNA amplification kit according to manufacturer's recommendations, but both protocols were modified to include a decrosslinking step of an overnight (14-16 hours) incubation at 65°C. After the whole-genome amplification procedure, the DNA was purified. 1 μg (or all available amount, if the yield was lower) of DNA was diluted to 500 μl with sonication buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1% SDS) and was sonicated using Branson Sonifier 150 for 20 seconds with power setting 3. The sonicated DNA was purified and then submitted for library preparation and sequencing.
The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample  preparation protocol for Solexa platforms (NEB reagents).

tag: strain


title: 43_oocyte_Intermediate


title: 41_oocyte_NSN


title: 42_oocyte_SN-Hoechst


title: HER2 Cas9 sat A431 High Rep 1

city: Durham

first: Charles

middle: A

last: Gersbach

state: North Carolina

address: 101 science drive rm 2323

country: USA

institute: Duke University

data_processing:
Library strategy: CRISPR/Cas9 Epigenentic Regulatory Element Screening (CERES)
FASTQ files were aligned to custom indexes (generated from the bowtie2-build function) using Bowtie2 (v2.2.4) with the options -p 32 --end-to-end --very-sensitive -3 1 -I 0 -X 200.
Reads were converted from .sam to .bam using samtools view -bS (v1.3.1).
Sort reads using samtools sort
Index reads using samtools index
Reads were counted using samtools idxstats
Genome_build: hg19
Supplementary_files_format_and_content: Counts of the number of reads aligned to each gRNA sequence (txt format).

organism: Homo sapiens

source_name: A431 Cells

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO64

extract_protocol:
An aliquot of 1.3x10^6 cells was taken before sorting for a bulk unsorted sample for each screen. The highest and lowest 10% of cells were sorted based on mCherry signal for the dCas9KRAB screen of HBE1 in K562 cells, or Alexa Fluor 488 signal for all HER2 screens. 1.3x10^6 cells were sorted for each group. Cell sorting was performed using a MoFlo Astrios (Beckman Coulter) or SH800 (Sony Biotechnology). After sorting, cells were harvested for genomic DNA using the DNeasy Blood and Tissue Kit (Qiagen).
To amplify the gRNA libraries from each sample, 8.3 µg of gDNA was used as template across 8 100 µL PCR reactions using Q5 hot start polymerase (NEB). Amplification was carried out following the manufacturer’s instructions using 25 cycles at an annealing temperature of 60C. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter) using double size selection of 0.65X and then to 1X the original volume. Each sample was quantified after purification using the Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher).

treatment_protocol: The A431-Cas9 cell line was derived by transducing A431 cells with a lentivirus containing Cas9-P2A-puromycin and, two days after transduction, selected with puromycin for 7 days.

tag: cell line

tag: transduced gene

tag: grna library

value: HER2 Saturation Library

tag: endogenous reporter


title: HER2 Cas9 sat A431 Low Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO63


title: HER2 Cas9 sat A431 Low Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO62


title: 49_oocyte_SN-Hoechst


title: 53_pronucleus_w-o-inh-both

source_name: zygote

growth_protocol: 3-4-week-old C57BL/6J females were superovulated by intraperitoneal injection of PMSG (pregnant mare's serum gonadotropin; 5 IU, Folligon, Intervet) followed by hCG (human chorionic gonadotropin; 5 IU, Chorulon, Intervet) injection 48 hours later. Natural matings to C57BL/6J males were set up overnight. Zygotes were released from the ampullae and treated with hyaluronidase (Sigma) to remove surrounding cumulus cells. For isolation of maternal and paternal nuclei, zygotes were pre-incubated in culture medium supplemented with 5 μg/ml cytochalasin B (Sigma) and 1 μM nocodazole (Sigma) for 15 minutes. The zona pellucida of the zygotes was opened and nuclei were extracted one by one. The maternal and paternal origin of the nuclei was determined taking size of the nuclei and position in relation to second polar body into account.

extract_protocol:
Nuclei were fixed in separate drops of M2 (not containing BSA) with 2% formaldehyde for 15 minutes. Nuclei were washed through drops of complete M2. For zygote experiments without pronuclear extraction, zygotes were treated identically to oocytes. Here nuclei were separated into individual wells after SDS treatment and kept separate in the subsequent steps of the protocol. After fixation, zygotes or single isolated pronuclei were incubated in 9 μl of lysis buffer for at least 15 minutes on ice. Similarly zygotes or pronuclei were incubated in 9 μl of 1× NEB3 buffer supplemented with 0.6% SDS for 2 hours at 37° with shaking in humidified atmosphere. Then nuclei were incubated in 1× DpnII buffer (NEB) with 1× BSA and  5 U DpnII (NEB). After overnight incubation in a humidified incubator at 37° the DNA ends were ligated in 9 μl of 1× T4 DNA ligase buffer containing 5 U T4 DNA ligase (Thermo Scientific) and they were incubated at 16°C for 4.5 hours with 50 rpm rotation, and then were kept for 30 min at room temperature. DNA from each nucleus was amplified using illustra GenomiPhi v2 DNA amplification kit (GE Healthcare) or illustra Single Cell GenomiPhi DNA amplification kit according to manufacturer's recommendations, but both protocols were modified to include a decrosslinking step of an overnight (14-16 hours) incubation at 65°C. After the whole-genome amplification procedure, the DNA was purified. 1 μg (or all available amount, if the yield was lower) of DNA was diluted to 500 μl with sonication buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1% SDS) and was sonicated using Branson Sonifier 150 for 20 seconds with power setting 3. The sonicated DNA was purified and then submitted for library preparation and sequencing.
The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample  preparation protocol for Solexa platforms (NEB reagents).


title: HER2 Cas9 sat A431 Low Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO61


title: 39_oocyte_SN


title: HER2 Cas9 sat A431 Unsorted Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO59


title: 45_pronucleus_w-o-inh-undef sex


title: 37_oocyte_SN


title: HER2 Cas9 sat A431 Unsorted Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO57


title: 151_pronucleus_female


title: 38_pronucleus_w-o-inh-undef sex


title: HER2 Cas9 sat A431 Unsorted Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO56


title: HER2 Cas9 sat A431 Unsorted Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO58


title: 101_oocyte_SN


title: HER2 p300 sat 293T High Rep 4

source_name: HEK293T Cells

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO58

treatment_protocol: The HEK293T-dCas9-p300 cell line was derived by transducing HEK293T cells with a lentivirus containing dCas9p300-P2A-puromycin and, two days after transduction, selected with puromycin for 7 days.


title: HER2 p300 sat 293T High Rep 3

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO57


title: 34_oocyte_SN-Hoechst


title: HER2 p300 sat 293T High Rep 2

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO56


title: 35_oocyte_SN


title: 30_oocyte_NSN


title: 33_oocyte_SN-Hoechst


title: 31_oocyte_SN


title: 32_oocyte_NSN


title: 29_oocyte_SN


title: 27_oocyte_SN


title: HER2 p300 sat 293T Low Rep 3

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO53


title: HER2 p300 sat 293T Low Rep 1

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO51


title: HER2 p300 sat 293T Low Rep 4

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO54


title: 28_oocyte_SN-Hoechst


title: 25_oocyte_NSN


title: 36_pronucleus_w-o-inh-both


title: HER2 p300 sat 293T Unsorted Rep 4

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO50


title: 23_oocyte_SN-Hoechst

growth_protocol: Fully grown denuded GV oocytes were isolated from 2-3 month old C57BL/6J mice by puncturing of ovaries with hypodermic needles28. Oocytes were incubated for at least 2 hours before the procedure to ensure viability. Before formaldehyde fixation, the zona pellucida was removed by treatment with Acidic Tyrode's solution (Sigma).


title: HER2 p300 sat 293T High Rep 1

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO55


title: 24_pronucleus_w-o-inh-undef sex


title: HER2 p300 sat A431 High Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO55

treatment_protocol: The A431-dCas9-p300 cell line was derived by transducing A431 cells with a lentivirus containing dCas9p300-P2A-puromycin and, two days after transduction, selected with puromycin for 7 days.


title: HER2 p300 sat 293T Low Rep 2

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO52


title: HER2 p300 sat A431 High Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO54


title: 20_oocyte_SN


title: 21_oocyte_SN-Hoechst


title: 19_oocyte_SN-Hoechst


title: HER2 p300 sat A431 Low Rep 4

data_processing:
Library strategy: CRISPR/Cas9 Epigenentic Regulatory Element Screening (CERES)
FASTQ files were aligned to custom indexes (generated from the bowtie2-build function) using Bowtie2 (v2.2.4) with the options -p 32 --end-to-end --very-sensitive -3 1 -I 0 -X 200.
Reads were converted from .sam to .bam using samtools view -bS (v1.3.1).
Sort reads using samtools sort
Index reads using samtools index
Reads were counted using samtools idxstats
Genome_build: hg19
Supplementary_files_format_and_content: Counts of the number of reads aligned to each gRNA sequence.

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO52


title: HER2 p300 sat A431 High Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO53


title: HER2 p300 sat A431 Low Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO51


title: 17_oocyte_NSN


title: 18_oocyte_SN-Hoechst


title: 47_oocyte_NSN


title: 14_oocyte_SN


title: 16_oocyte_SN-Hoechst


title: HER2 p300 sat A431 Unsorted Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO48


title: HER2 p300 sat 293T Unsorted Rep 2

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO48


title: HER2 p300 sat A431 Low Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO49


title: HER2 p300 sat A431 Unsorted Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO47


title: 12_oocyte_NSN


title: HER2 p300 sat 293T Unsorted Rep 1

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO47


title: HER2 p300 sat A431 Low Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO50


title: 22_oocyte_SN-Hoechst


title: HER2 p300 sat A431 High Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO56


title: HER2 p300 sat 293T Unsorted Rep 3

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO49


title: 26_oocyte_NSN-Hoechst


title: HER2 p300 A431 High Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO44

value: HER2 DHS Library


title: HER2 p300 sat A431 Unsorted Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO45


title: 11_oocyte_SN-Hoechst

growth_protocol: Fully grown denuded GV oocytes were isolated from 2-3 month old C57BL/6J mice by puncturing of ovaries with hypodermic needles28. Oocytes were incubated for at least 2 hours before the procedure to ensure viability. Before formaldehyde fixation, the zona pellucida was removed by treatment with Acidic Tyrode's solution (Sigma).


title: 10_oocyte_NSN


title: HER2 p300 A431 High Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO43


title: 8_oocyte_SN


title: HER2 p300 A431 Low Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO40


title: 9_oocyte_SN-Hoechst


title: 3_oocyte_SN


title: 7_oocyte_Intermediate


title: HER2 p300 A431 Unsorted Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO38


title: HER2 p300 A431 Low Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO39


title: 5_oocyte_NSN


title: 6_oocyte_SN-Hoechst


title: HER2 p300 A431 Unsorted Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO37


title: HER2 p300 sat A431 Unsorted Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO46


title: HER2 p300 A431 Unsorted Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO36


title: HER2 p300 293T High Rep 4


title: HER2 p300 293T High Rep 2

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO45


title: HER2 p300 293T High Rep 3

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO46


title: 4_oocyte_NSN


title: 1_oocyte_NSN


title: HER2 p300 293T Low Rep 4

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO43


title: HER2 p300 293T High Rep 1

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO44


title: 13_oocyte_SN


title: HER2 p300 293T Low Rep 3

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO42


title: HER2 p300 A431 High Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO42


title: HER2 p300 293T Low Rep 1

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO40


title: HER2 p300 293T Unsorted Rep 4

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO39


title: HER2 p300 293T Unsorted Rep 3

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO38


title: HER2 p300 293T Unsorted Rep 2

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO37


title: 129SvJ F IMQ 2

city: Ann Arbor

first: William

middle: R

last: Swindell

state: MI

address: 1500 E. Medical Center Drive

department: Dermatology

institute: University of Michigan

description: RNA extracted from full-thickness skin biopsy (IMQ-treated)

library_source: transcriptomic

data_processing:
Illumina adaptor sequences were removed using CutAdapt (version 1.6) with a maximum error rate setting (-e) of 5% and a minimum post-processing read length (-m) of 20 base pairs
Reads were trimmed to remove bases with low PHRED quality scores (CutAdapt and Fast-X toolkit)
Filtered reads were mapped to the UCSC mouse genome (mm10) using Tophat2 (version 2.0.12)
Overlap between mapped reads and known human genes was tabulated using the htseq-count function from the HTSeq python library (version 0.5.4p3)
Cufflinks (version 2.2.1) was used to calculate fragments per kilobase of exon per million fragments mapped (FPKM) for each gene feature.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data includes raw read counts and FPKM values for all mouse genes (UCSC, mm10) and each sample (.txt files).

molecule: total RNA

source_name: skin

growth_protocol: Male and female mice were purchased at 8 weeks of age and allowed 14 days to acclimate prior to starting experiments. Most strains were purchased from the Jackson Laboratory (C57BL/6J, cat. no. 000664; BALB/cJ, cat. no. 000651; DBA/1J, cat. no. 000670; FVB/NJ, cat. no. 001800; 129X1/SvJ, cat. no. 000691; MOLF/EiJ, cat. no. 000550). CD1 (ICR) mice were purchased from Charles River laboratories (cat. no. 022). Mice were maintained within the Case Western Reserve University animal vivarium under specific-pathogen free conditions and provided standard rodent chow diet (4.5% fat by weight, 14% kcal, Lab Diet P3000).

extract_protocol:
Total RNA was extracted from flash-frozen skin samples using the RNeasy Fibrous Tissue Mini Kit (Qiagen, cat. no. 74704) with on-column DNase digestion (Qiagen, cat. no. 79254). RNA concentrations were evaluated using the NanoDrop spectrophotometer and RNA integrity was assessed using the Agilent Bioanalyzer. RNA was further assessed for quality using the TapeStation (Agilent, Santa Clara, CA) with manufacturer's recommended protocols. All samples had RNA integrity numbers of 8 or greater.
Sequencing of fragmented complementary cDNA was performed using the Illumina Genome Analyzer Iix

treatment_protocol: Mouse back skin was treated with 62.5 mg AldaraTM (5% IMQ) or a non-toxic lanolin-derived occlusion cream (CTL) once per day for 5 consecutive days. IMQ and CTL treatments were carried out for 5 consecutive days and mice were sacrificed on day 6 following injection with euthanasia solution (ketamine, 16.5mg/mL; xylazine, 1.65 mg/mL) followed by cervical dislocation.

tag: Sex

value: female

tag: treatment

value: IMQ


title: 2_oocyte_SN-Hoechst

growth_protocol: Fully grown denuded GV oocytes were isolated from 2-3 month old C57BL/6J mice by puncturing of ovaries with hypodermic needles28. Oocytes were incubated for at least 2 hours before the procedure to ensure viability. Before formaldehyde fixation, the zona pellucida was removed by treatment with Acidic Tyrode's solution (Sigma).


title: HER2 p300 293T Unsorted Rep 1

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO36


title: 129SvJ F CTL 2

description: RNA extracted from full-thickness skin biopsy (control-treated)

value: control


title: HER2 KRAB A431 day 11 High Rep 4

treatment_protocol: The A431-dCas9-KRAB cell line was derived by transducing A431 cells with a lentivirus containing dCas9-KRAB PGK-HygromycinR in media containing 4 µg/mL polybrene and, two days after transduction, selected with Hygromycin B (200 µg/mL) for 7 days.


title: HER2 p300 A431 Low Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO41


title: HER2 KRAB A431 day 11 High Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO35


title: 129SvJ M IMQ 2

value: male


title: HER2 KRAB A431 day 11 High Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO34


title: 129SvJ M IMQ 1


title: HER2 KRAB A431 day 11 High Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO33


title: HER2 KRAB A431 day 11 Low Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO31


title: HER2 KRAB A431 day 11 Low Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO32


title: HER2 p300 293T Low Rep 2

growth_protocol: HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO41


title: HER2 KRAB A431 day 11 Unsorted Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO28


title: 15_oocyte_NSN


title: HER2 KRAB A431 day 11 Unsorted Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO27


title: HER2 KRAB A431 day 11 Low Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO29


title: 129SvJ M CTL 1


title: HER2 KRAB A431 day 11 Unsorted Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO26


title: HER2 KRAB A431 day 11 Low Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO30


title: MOLF F IMQ 2

value: MOLF/EiJ


title: 129SvJ F IMQ 1


title: HER2 KRAB A431 day 19 High Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO24


title: HER2 KRAB A431 day 11 Unsorted Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO25


title: HER2 KRAB A431 day 19 High Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO23


title: MOLF F IMQ 1

growth_protocol: Male and female mice were purchased at 8 weeks of age and allowed 14 days to acclimate prior to starting experiments. Most strains were purchased from the Jackson Laboratory (C57BL/6J, cat. no. 000664; BALB/cJ, cat. no. 000651; DBA/1J, cat. no. 000670; FVB/NJ, cat. no. 001800; 129X1/SvJ, cat. no. 000691; MOLF/EiJ, cat. no. 000550). CD1 (ICR) mice were purchased from Charles River laboratories (cat. no. 022). Mice were maintained within the Case Western Reserve University animal vivarium under specific-pathogen free conditions and provided standard rodent chow diet (4.5% fat by weight, 14% kcal, Lab Diet P3000).


title: 129SvJ F CTL 1


title: HER2 KRAB A431 day 19 Low Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO20


title: 129SvJ M CTL 2


title: 66_pronucleus_female

growth_protocol: 3-4-week-old C57BL/6J females were superovulated by intraperitoneal injection of PMSG (pregnant mare's serum gonadotropin; 5 IU, Folligon, Intervet) followed by hCG (human chorionic gonadotropin; 5 IU, Chorulon, Intervet) injection 48 hours later. Natural matings to C57BL/6J males were set up overnight. Zygotes were released from the ampullae and treated with hyaluronidase (Sigma) to remove surrounding cumulus cells. For isolation of maternal and paternal nuclei, zygotes were pre-incubated in culture medium supplemented with 5 μg/ml cytochalasin B (Sigma) and 1 μM nocodazole (Sigma) for 15 minutes. The zona pellucida of the zygotes was opened and nuclei were extracted one by one. The maternal and paternal origin of the nuclei was determined taking size of the nuclei and position in relation to second polar body into account.


title: MOLF F CTL 2


title: MOLF F CTL 1


title: HER2 KRAB A431 day 19 High Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO21


title: HER2 KRAB A431 day 19 Low Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO18


title: MOLF M IMQ 2


title: HER2 KRAB A431 day 19 Low Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO19


title: HER2 KRAB A431 day 19 Unsorted Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO14


title: HER2 KRAB A431 day 19 Low Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO17


title: HER2 KRAB A431 day 19 Unsorted Rep 4

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO16


title: HER2 KRAB A431 day 19 Unsorted Rep 1

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO13


title: HER2 KRAB A431 day 19 Unsorted Rep 3

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO15


title: MOLF M CTL 2


title: MOLF M IMQ 1


title: HBE1 KRAB K562 High Rep 4

source_name: K562 Cells

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO13

treatment_protocol: The K562-dCas9KRAB HBE1 reporter cell line was generated by electroporation of 2 x 106 cells with a donor plasmid (6.67 µg), a HBE1 stop codon-targeting gRNA plasmid (6.67 µg), and a Cas9 plasmid (6.67 µg), as described previously3. Transfected cells were selected with puromycin (Sigma, P8833) at a concentration of 1 µg/mL, beginning 2 days after electroporation, for 7 days. To remove the puromycin selection cassette, cells were transfected with a CMV-CRE plasmid (20 µg) and cultured for 10 days without antibiotic selection. Next, the cells were transduced with a lentivirus containing dCas9KRAB in media containing 4 µg/mL polybrene (Santa Cruz, sc-134220). Cells were selected with Hygromycin B (200 µg/mL) for 7 days. Cells were then clonally isolated via serial dilutions to obtain a single clone harboring the knocked-in reporter without the puromycin cassette and dCas9KRAB.

value: HBE1 DHS Library


title: DSCP_500bp_dmel_input

city: Vienna

first: Alexander

last: Stark

address: Campus-Vienna-Biocenter 1

country: Austria

institute: The Research Institute of Molecular Pathology (IMP)

description:
PCR amplified Plasmid library
processed data file:

data_processing:
library strategy: STARR-Seq
Reads were mapped to the dm3 genome using bowtie (bowtie -p 12 -f -X 2000 -v 3 -m 1 --best --strata --quiet INDEX -1 reads_1.fa -2 reads_2.fa) excluding chrU, chrUextra, and chrM. For all subsequently analysis we used only reads collapsed on chromosome, start, end, strand (fragments). Significantly enriched regions (peaks) were called with an in-house pipeline using read density profiles of cDNA and input and an hypergeometric test to assign a p-value to each peak. Enrichment values were corrected within a 95% confidence interval taking into account the number of independent fragments at a single peak summit position.
Genome_build: dm3
Supplementary_files_format_and_content: All processed data files are in plain text. For STARR-seq we report for each peak the chromosome, the summit position, the enrichment over input (log2-scale) at the summit, and the p-value.

organism: Drosophila melanogaster

growth_protocol: Drosophila Kc167 cell lines were cultured in M3+BYPE medium, supplemented with 5% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 25°C.

extract_protocol:
In 24 hrs cells were harvested and polyA RNA was isolated as in Shlyueva et al. Molecular Cell. 2014.
Total RNA was reverse transcribed and STARR-seq vectors were amplified from cDNA as described in Arnold et al. Science. 2013

treatment_protocol: After electroporation of the STARR-seq screening library, wild type Drosophila Kc167 cells or Pangolin mutated Drosophila Kc167 cells were treated with 25µM CHIR99021 or DMSO for 24h. STARR-seq screening library was made as described in Shlyueva et al. Molecular Cell. 2014. In brief: Genomic DNA (source: embryos of the sequenced strain: y; cn bw sp) was isolated, sheared and size selected (~500bp). Following the instructions of NEBNext® DNA Library Prep Reagent Set for Illumina® (NEB; cat. no. E6000L), Illumina Multiplexing Adapters (Illumina Inc; cat. no. PE-400-1001) were ligated and homology arms for In-Fusion® recombination were added by PCR, followed by recombination into the STARR-seq vector (pGL3-Promotor backbone (Promega; cat. no. E1751) with the sequence between BglII and FseI replaced with the following sequence, containing a Drosophila Synthetic Core Promoter (DSCP) (1), an ORF (sgGFP, Qbiogene, Inc), a ccdB suicide gene flanked by homology arms (used for cloning the genomic enhancer candidates during library generation), and the pGL3’s SV40 late polyA-signal.). The In-Fusion® reactions were transformed (MegaX DH10B; Invitrogen), grown in liquid culture and plasmids were isolated.

tag: molecule subtype

value: plasmid DNA (containing genomic DNA fragments)


title: HBE1 KRAB K562 High Rep 3

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO12


title: MOLF M CTL 1


title: KcWT_STARR_inh_rep2

description:
PCR amplified cDNA (STARR-seq transcript)
processed data file:
KcWT_STARR_inh.merged.pe.peaks.txt
KcWT_STARR_DSCP_DMSO-inh.pe.evalPosw250.txt

molecule: polyA RNA

tag: cell type

value: Kc167 cells

tag: genotype/variation

value: wild type

tag: treated with


title: FVB F IMQ 2

value: FVB/NJ


title: HBE1 KRAB K562 High Rep 1

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO10


title: HBE1 KRAB K562 High Rep 2

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO11


title: HBE1 KRAB K562 Low Rep 4

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO9


title: KcWT_STARR_inh_rep1


title: HER2 KRAB A431 day 19 High Rep 2

growth_protocol: A431 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM High Glucose supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO22


title: KcWT_STARR_DMSO_rep2

description:
PCR amplified cDNA (STARR-seq transcript)
processed data file:
KcWT_STARR_DMSO.merged.pe.peaks.txt
KcWT_STARR_DSCP_DMSO-inh.pe.evalPosw250.txt

value: DMSO


title: HBE1 KRAB K562 Low Rep 2

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO7


title: HBE1 KRAB K562 Unsorted Rep 4

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO5


title: FVB F IMQ 1

growth_protocol: Male and female mice were purchased at 8 weeks of age and allowed 14 days to acclimate prior to starting experiments. Most strains were purchased from the Jackson Laboratory (C57BL/6J, cat. no. 000664; BALB/cJ, cat. no. 000651; DBA/1J, cat. no. 000670; FVB/NJ, cat. no. 001800; 129X1/SvJ, cat. no. 000691; MOLF/EiJ, cat. no. 000550). CD1 (ICR) mice were purchased from Charles River laboratories (cat. no. 022). Mice were maintained within the Case Western Reserve University animal vivarium under specific-pathogen free conditions and provided standard rodent chow diet (4.5% fat by weight, 14% kcal, Lab Diet P3000).


title: FVB F CTL 2


title: FVB F CTL 1


title: HBE1 KRAB K562 Low Rep 3

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO8


title: HBE1 KRAB K562 Unsorted Rep 3

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO4


title: HBE1 KRAB K562 Low Rep 1

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO6


title: STARR_PanKO_inh_rep2

description:
PCR amplified cDNA (STARR-seq transcript)
processed data file:
STARR_PanKO_inh.merged.pe.peaks.txt
STARR_PanKO_DMSO-inh.pe.evalPosw250.txt

value: Pangolin mutated


title: HBE1 KRAB K562 Unsorted Rep 1

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO2


title: STARR_PanKO_inh_rep1


title: HBE1 KRAB K562 Unsorted Rep 2

growth_protocol: K562 cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO3


title: STARR_PanKO_DMSO_rep1

description:
PCR amplified cDNA (STARR-seq transcript)
processed data file:
STARR_PanKO_DMSO.merged.pe.peaks.txt
STARR_PanKO_DMSO-inh.pe.evalPosw250.txt


title: A3302G

city: Perth

first: Stefan

middle: J

last: Siira

state: Western Australia

address: 15 Hospital Ave, Nedlands

department: Precision Health

country: Australia

institute: The Kids Research Institute Australia

description: Mitochondrial RNA

data_processing:
Index sequences (5´-ATCACG-3´ and 5´-CGATGT-3´ for m.3243A>G & m.3302A>G, respectively) were trimmed with cutadapt (-e 0.0 -n 1 -O 6 –quality-base=64).
Reads that were 20 or 21 nt in length after trimming were extracted with awk and aligned uniquely to the human mitochondrial genome (hg38) with Bowtie 1.1.2 (-yS -m 1 –phred64-quals).
Strand-specific 5´ end profiles normalised to library size (reads per million, RPM) were generated with BEDtools 2.25.0 (-d -5 -strand [-/+] -scale [1,000,000/total mapped reads]) and converted to bedGraph format for visualisation.
Genome_build: hg38
Supplementary_files_format_and_content: Strand-specific bedGraph files containing normalised 5’ end PARE tag abundance

source_name: 143B osteocarcinoma cells

growth_protocol: Cybrid cell lines were cultured at 37 °C under humidified 95% air/5% CO2 in Dulbecco's modified Eagle's medium (DMEM, Gibco, Life Technologies) containing glucose (4.5 g/l− 1), 1 mM pyruvate, 50 μg/ml uridine, 2 mM glutamine, penicillin (100 U/ml− 1), streptomycin sulfate (100 μg/ml− 1) and 10% fetal bovine serum (FBS).

extract_protocol:
Mitochondria were prepared from 107 cells grown overnight in 15 cm2 dishes and isolated as described previously (Rackham et al., Nucleic Acids Res. Vol.37 No.17, 2009), with some modifications. Mitochondria were lysed for 30 min in buffer containing 250 mM sucrose, 100 mM KCl, 20 mM magnesium acetate, 10 mM Tris–HCl pH 7.5, 0.5% Triton X-100 and EDTA-free Complete protease inhibitor cocktail (Roche). RNA was isolated from mitochondria using the miRNeasy Mini kit (Qiagen) incorporating an on-column RNase-free DNase digestion to remove all DNA.
Mitochondrial RNA was prepared according to the PARE protocol as described by German et al. (Nature Protocols, Vol.4 No.3, 2009). Raw reads were processed follows: 3´ adaptor sequences were trimmed with cutadapt 1.9.1 (-e 0.1 -n 2 -O 6 --quality-base=64 –match-read-wildcards -a TCGTATGCCGTCTTCTGCTTG), followed by library splitting by index sequence (5´-ATCACG-3´ and 5´-CGATGT-3´ for m.3243A>G & m.3302A>G, respectively) with fastx_barcode_splitter.pl 0.0.13 from the FASTX-Toolkit (-eol -exact).

tag: mtdna mutation


title: FVB M IMQ 1


title: STARR_PanKO_DMSO_rep2


title: A3243G

description: Mitochondrial RNA


title: FVB M CTL 2


title: DBA F IMQ 2


title: WT5_rep2

city: Napoli

first: Annamaria

last: Carissimo

address: via Castellino

country: Italy

institute: Tigem

description: mIRNA enriched-total RNA

data_processing:
reads were aligned both to human mature miRNAs (miRBase v.20), using the CASAVA software (Illumina).
Genome_build: miRBAse v.20
Supplementary_files_format_and_content: [.txt] tab-delimited text files include raw counts

source_name: WT5, liver

extract_protocol:
Livers were dissected and flash-freezen using liquid nitrogen. miRNA enriched-total RNA was extracted using miRNeasy kit (QIAGEN).
Small RNA libraries were constructed using a TruSeq small RNA sample preparation kit (Illumina). We generated small RNA-seq libraries using 1 μg of total RNA as input from each sample. By multiplexing up to 12 samples were combined into a single lane to obtain sufficient coverage and two technical replicates were run for each library

tag: tissue

value: liver

tag: age

value: 6 weeks

value: wild-type


title: FVB M CTL 1


title: WT5_rep1

description: mIRNA enriched-total RNA


title: FVB M IMQ 2


title: WT4_rep2

description: mIRNA enriched-total RNA

source_name: WT4, liver


title: DBA F CTL 2


title: WT3_rep2

description: mIRNA enriched-total RNA

source_name: WT3, liver


title: DBA F CTL 1


title: DBA F IMQ 1


title: WT4_rep1

description: mIRNA enriched-total RNA


title: WT3_rep1

description: mIRNA enriched-total RNA


title: WT2_rep2

description: mIRNA enriched-total RNA

source_name: WT2, liver


title: KcWT_STARR_DMSO_rep1


title: DBA M CTL 2


title: CD1 F IMQ 2


title: DBA M IMQ 2


title: DBA M CTL 1


title: CD1 F IMQ 1


title: DBA M IMQ 1


title: WT2_rep1

description: mIRNA enriched-total RNA


title: WT1_rep2

description: mIRNA enriched-total RNA

source_name: WT1, liver


title: WT1_rep1

description: mIRNA enriched-total RNA


title: piz6_272_rep1

description: mIRNA enriched-total RNA

source_name: piz6_272, liver

value: PiZ


title: CD1 M IMQ 2


title: CD1 F CTL 1


title: CD1 F CTL 2


title: piz6_269_rep2

description: mIRNA enriched-total RNA

source_name: piz6_269, liver


title: piz6_271_rep2

description: mIRNA enriched-total RNA

source_name: piz6_271, liver


title: piz6_269_rep1

description: mIRNA enriched-total RNA


title: piz6_271_rep1

description: mIRNA enriched-total RNA


title: CD1 M IMQ 1


title: piz6_115_rep1

description: mIRNA enriched-total RNA

source_name: piz6_115, liver


title: CD1 M CTL 2


title: 281116_A_S1

city: Lausanne

first: Evarist

last: Planet

state: Vaud

address: Route Cantonale

institute: EPFL

description: TI ChIP hESC

data_processing:
Alignement for ChIP-seqs were done with bowtie2: bowtie2 -p 6 -t --sensitive-local -x $index -1 $reads1 -2 $reads2
Peak calling for ChIP-seqs were done using MACS2: macs2 callpeak -t $chipbam -c $tibam -f BAM -g $org -n $name -B -q 0.01 --format BAMPE
Genome_build: hg19
Supplementary_files_format_and_content: A bed file containing the output of the peak called by MACS2 is provided.

source_name: H1 human embryonic stem cells

growth_protocol: H1 human embryonic stem cells were cultured in mTesRI on hES-qualified Matrigel

extract_protocol:
Total inputs and ChIPed samples were prepared for libraries using 10ng of material. Aliquots of the samples were tested in qPCR using the KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems, KK4604) to determine the optimal number of PCR cycles needed to amplify each library without reaching saturation. Libraries were amplified with KAPA HiFi Hotstart Readymix polymerase (KAPA Biosystems, KK2602), size-selected using Ampure XP beads (Beckman Coulter) and always quality checked on Bioanalyser DNA high sensitivity chip (Agilent) and quantified by picogreen (Qubit® 2.0 Fluorometer, Invitrogen).
Chromatin was prepared from 10 million cells as previously described (Rowe et al., 2013b).

treatment_protocol: Cells were transfected with DUX4 using the TransIT transfection reagent. Fixation was done 24h after transfection

tag: source

value: H1 human embryonic stem cells (hESCs)

tag: transfected with

value: plasmid encoding for a HA-tagged form of DUX4

tag: time point

value: 24 hours after transfection

tag: chip antibody


title: BalbC F IMQ 1

tag: strain

value: BALB/cJ

value: female

value: IMQ


title: BalbC F IMQ 2


title: CD1 M CTL 1

value: male

value: control


title: piz6_115_rep2

description: mIRNA enriched-total RNA


title: 021216_E_S4

description: DUX4 ChIP hESC

extract_protocol:
Libraries were ligated with Illumina adaptors
Chromatin was prepared from 10 million cells as previously described (Rowe et al., 2013b).


title: BalbC M IMQ 1


title: piz6_272_rep2

description: mIRNA enriched-total RNA


title: BalbC M CTL 2


title: BalbC M CTL 1


title: B6 F IMQ 2


title: 232_K562-A

source_name: chronic myelogenous leukemia cells

growth_protocol: K562 (a gift from Alexander Stark lab) cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-Glutamine and Pen/Strep (100 U/ml and 100 μg/ml).

extract_protocol:
2-4 million cells were fixed for 10 minutes by resuspending the cell pellet in 5 ml full culture medium supplemented with 1% formaldehyde (Sigma). The reaction was quenched by addition of 2 M glycine to a final concentration of 125 mM and incubation for 5 minutes on ice. Cells were pelleted by centrifugation, washed by resuspension in 1 ml of PBS and pelleted again. Then the cells were resuspended in 1.5 ml of lysis buffer and incubated on ice for at least 15 minutes. Cells were pelleted by centrifugation at 2,500g for 5 minutes, resuspended in 100 μl of 1×NEBuffer 3 and pelleted again. The pellet was resuspended in 100 μl of 0.3% SDS in 1×NEBuffer 3 and incubated at 37°C for 1 hour. Then the suspension was diluted with 330 μl of 1× NEBuffer 3 and 53 μl of 20% Triton X-100 (Sigma), and incubated at 37°C for 1 hour to quench SDS. The cells were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 250 μl of 1× DpnII buffer (NEB). 20 μl were taken as a chromatin integrity control, then 600 U DpnII enzyme (NEB) were added, and the chromatin was digested overnight at 37°C with rotation. In the morning 200 more units of DpnII were added and the cells were incubated for additional 2 hours. DpnII was inactivated by incubation at 65°C for 20 minutes. 20 μl was taken as a digestion control. Nuclei were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 100 μl of 1× T4 DNA ligase buffer (Thermo Scientific) and pelleted again. The pellet was resuspended in 500 μl of 1× T4 DNA ligase buffer, and 50 U T4 DNA ligase (Thermo Scientific) were added. The sample was mixed by inversion and incubated with rotation for 4 hours at 16°C and then for 30 minutes at room temperature. Then the nuclei were pelleted by centrifugation at 3,000g for 5 minutes and resuspended in 120 μl of sterile PBS. 20 μl were taken as a ligation control. The rest of the sample was stained with Hoechst, single G1 nuclei were sorted using excitation wavelength 375 nm and forward and side scatter by FACS (FACSAriaIII machine, BD Biosciences) into wells of 12 PCR strips containing 3 μl sample buffer from illustra GenomiPhi v2 DNA amplification kit (GE Healthcare), then they were covered by 5 μl of mineral oil, incubated at 65°C overnight and amplified according to a protocol from Kumar et al, 2008. The amplification was considered positive if the sample contained ≥1 μg DNA, and these samples were submitted for library preparation and sequencing
The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample  preparation protocol for Solexa platforms (NEB reagents).


title: BalbC F CTL 1


title: 242_K562-A


title: B6 F CTL 1


title: B6 F CTL 2


title: piz6_107_rep1

description: mIRNA enriched-total RNA

source_name: piz6_107, liver


title: BalbC F CTL 2


title: 241_K562-A


title: 240_K562-A


title: B6 M IMQ 2


title: 238_K562-A


title: 235_K562-A


title: piz6_107_rep2

description: mIRNA enriched-total RNA


title: 229_K562-A


title: B6 M CTL 1


title: B6 M CTL 2


title: 233_K562-A


title: 234_K562-A


title: BalbC M IMQ 2


title: 226_K562-B


title: 236_K562-A


title: 237_K562-A


title: B6 M IMQ 1


title: LaptTA-fos-MUT_2mo_2

first: Rainer

last: Hamacher

address: Waehringer Gürtel 18-20

department: Dept. for Internal Medicine I / Oncology

institute: Medical University Vienna - AKH

description:
Isolation of poly A+ RNA from total RNA
Strand-specific RNA-seq (dUTP)

data_processing:
Reads were aligned to the mouse genome (GRCm38/mm10) with TopHat-2.0.4 (using Bowtie 0.12.7 and Samtools 0.1.16), allowing 2 mismatches and 5 multihits
Transcripts quantification and differential expression were calculated with Cufflinks 1.3.0, using the Mus musculus GRCm38/mm10 transcript annotations from the UCSC Genome Browser
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: FPKM values calculated with Cufflinks

source_name: liver tissue obtained from our mutant mouse (LAP-tTA; col1a1:Tet-O-fosFlag ) at 2 months hepatocyte-specific c-fos expression by removal of doxycycline at the age of 3 weeks

extract_protocol:
RNA was isolated using Trizol (Sigma-Aldrich) according to the manufacturer’s instructions
RNA processing was performed as described in Illuminas “TruSeq RNA Sample Preparation V2” (Part #15026495 Rev B February 2012)

treatment_protocol: Doxycycline (1 g/l) was supplied in sucrose-containing (50 g/l) drinking water or food pellets. 100mg/kg Diethylnitrosamine (DEN) was injected intraperitoneal (i.p.).

tag: background strain

value: doxycycline from conception until 3 weeks postnatal

tag: genotype

value: LAP-tTA; col1a1:Tet-O-fosFlag


title: 231_K562-A


title: LaptTA-fos-CO_2mo_2

source_name: liver tissue obtained from our single-transgenic control mouse (LAP-tTA; col1a1:Tet-O-fosFlag ) at 2 months after removal of doxycycline at the age of 3 weeks

value: single transgenic control LAP-tTA; col1a1:Tet-O-fosFlag


title: 228_K562-B


title: 239_K562-A


title: LaptTA-fos-CO_2mo_1


title: LaptTA-fos-MUT_4mo_3

source_name: liver tissue obtained from our mutant mouse (LAP-tTA; col1a1:Tet-O-fosFlag ) at 4 months hepatocyte-specific c-fos expression by removal of doxycycline at the age of 3 weeks


title: LaptTA-fos-MUT_4mo_1


title: LaptTA-fos-CO_4mo_3

source_name: liver tissue obtained from our single-transgenic control mouse (LAP-tTA; col1a1:Tet-O-fosFlag ) at 4 months after removal of doxycycline at the age of 3 weeks


title: 230_K562-A


title: LaptTA-fos-CO_4mo_2


title: LaptTA-fos-MUT_4mo_2


title: LaptTA-fos-CO_4mo_1


title: 225_K562-A


title: AlfpCre-fos-MUT_DEN_2

description:
Isolation of poly A+ RNA from total RNA
Unstranded RNA-seq

source_name: liver tissue obtained from our 8 week old mutant mouse (c-fos f/f; Alfp-Cre wt/T ) 48h after diethylnitrosamine injection 100mg/kg

value: one i.p. injection diethylnitrosamine 100mg/kg

value: c-fos f/f; Alfp-Cre wt/T


title: 227_K562-A


title: LaptTA-fos-MUT_2mo_1

value: liver


title: AlfpCre-fos-MUT_DEN_3


title: 222_K562-B


title: AlfpCre-fos-MUT_DEN_1


title: AlfpCre-fos-CO-Cre+_DEN_3

source_name: liver tissue obtained from our 8 week old control mouse (c-fos wt/wt; Alfp-Cre wt/T ) 48h after diethylnitrosamine injection 100mg/kg

value: c-fos wt/wt; Alfp-Cre wt/T


title: AlfpCre-fos-CO-Cre+_DEN_1


title: AlfpCre-fos-CO-Cre+_DEN_2


title: 224_K562-B


title: 220_K562-B


title: 217_K562-B


title: MEL cells 2% 15 min fixation

source_name: murine erythroleukemia, erythroid progenitor cells

growth_protocol: MEL cells (murine erythroleukemia cell line; ATCC) were cultured in RPMI-1640 medium supplemented with FBS and Pen/Strep (100 U/ml and 100 μg/ml)

extract_protocol:
7.5 million cells were fixed for 10 or 15 minutes by resuspending cell pellet in 7.5 ml full culture medium supplemented with 1% or 2% formaldehyde (Sigma), respectively. The reaction was quenched by addition of 2M glycine to a final concentration of 0.2 M and incubation for 5 minutes at room temperature on a rocker. Cells were pelleted by centrifugation at 300 g for 5 minutes at 4°C, washed by resuspension in 1 ml of PBS, pelleted and resuspended in 1 ml of PBS again. 1.9 mln cells from each sample were taken, the rest were snap-frozen for other experiments. The samples were pelleted again, then resuspended in 300 μl of cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40 substitute (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) and incubated on ice for at least 15 minutes. Cells were pelleted by centrifugation at 2,500g for 5 minutes, resuspended in 500 µl of lysis buffer and pelleted again. The pellet was resuspended in 50 μl 0.3% SDS in NEBuffer 3 and incubated at 62°C for 10 minutes. Then the suspension was diluted with 145 μl of 1× NEBuffer 3, 12.5 μl mQ and 12.5 μl of 20% Triton X-100 and incubated at 37°C for 1 hour to quench SDS. 30 µl were taken as a chromatin integrity control. The cells were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 250 μl of 1× DpnII buffer (NEB) with 600 U DpnII enzyme (NEB), and the chromatin was digested overnight (14-16 hours) at 37°C with rotation. In the morning 200 more units were added and the cells were incubated for additional 2 hours. DpnII was inactivated by incubation at 65°C for 20 minutes. 40 μl was taken as a digestion control, 40 μl of mQ were added to the sample. The ends were filled-in by addition of 50 μl fill-in mix (36 ul mQ, 1.5 μl of 10 mM biotin-14-CTP, 1.5 μl of 10 mM dATP, 1.5 μl of 10 mM dGTP, 1.5 μl of 10 mM dTTP, 8 μl of 5 U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB)) and incubation at 37°C for 1.5 hours. Then 900 μl of ligation mix (712 μl water, 120 μl 10× NEB T4 DNA ligase buffer (NEB), 50 μl 20% Triton X-100, 12 μl 10 mg/ml BSA (NEB), 6 μl 5 U/μl T4 DNA Ligase (Thermo Scientific)) were added to the sample. The sample was mixed by inversion and incubated with rotation for 4 hours at room temperature. Then the nuclei were pelleted by centrifugation at 3,000g for 10 minutes and resuspended in 200 μl mQ. Proteins were degraded by addition of 15 μl of 20 mg/ml proteinase K and 20 μl of 10% SDS and incubation at 55°C for 30 minutes. Then 130 μl 5M NaCl were added and the samples were incubated at 65°C overnight (14-16 hours). The DNA was ethanol precipitated and then dissolved in 500 μl 10 mM Tris-HCl pH 8.0, washed twice on Amicon 30K filter units (Millipore) with 10 mM Tris-HCl pH 8.0 and then eluted into a clean tube. The volume was adjusted to 500 μl with sonication buffer (50 mM Tris, 10 mM EDTA, 0.1% SDS) and the samples were sonicated using VirSonic 100 (Virtis) with power setting 15 for 15 seconds twice with 1 minute interval. Samples were washed twice again using Amicon 30K filter units (Millipore). All consecutive steps until PCR were performed in LoBind tubes (Eppendorf). 30 µl of 10 mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies) were prepared for biotin pull-down by washing with 80 µl of 1× TWB (1× Tween Washing Buffer, 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20). Then the beads were resuspended in 60 μl of 2× BB (2× Binding Buffer: 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) and added to the samples; the suspension was incubated for 15 minutes at room temperature with rotation. After that the beads were used for library preparation.
The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample  preparation protocol for Solexa platforms (NEB reagents).


title: MEL cells 1% 10 min fixation


title: 223_K562-B


title: cRNA Mrpp3 rep2

description: mitochondrial RNA

data_processing:
Adapter trimming was performed using cutadapt version 1.10 (cutadapt -m 20  -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG -A AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG)
Trimmed pair-end reads were merged by FLASH version 1.2.11 (flash R1.fastq R2.fastq -M 240)
Repetitive sequences within reads by applying Tandem Repeats Finder (TRF) version 4.09 (trf .fasta 2 7 7 80 10 50 200 -h -d). The TRF output file was converted from dat file format to fastq format with Python vesrion 2.7.6  and R version 3.3.1
Alignment to the mouse mitochondrial genome was performed using Bowtie 2 vesrion 2.2.9 (bowtie2 -x --sensitive-local). The soft-clipping mode was used for the alignment, the remaining unaligned parts were extracted based on CIGAR filed information of sam format with Python version 2.7.6 script. The  split parts of each read are called “left”, “middle”, and “right”. Retrieved “left” and “right” parts were aligned again to the mouse mitochondrial genome with bowtie2 version 2.2.9, but this time using an end-to-end mode (bowtie2 -x).
The coverage of combined data was found with BEDtools genomecov version 2.26.0  and count per million (CPM) normalized to the library size of all replicates of KO/WT. Intermediate steps were achieved using Python version 2.7.6 and R version 3.3.1.
Genome_build: mm10
Supplementary_files_format_and_content: bedgraph files were generated

source_name: heart

extract_protocol:
Mitochondria were isolated from homogenized hearts and purified by differential centrifugation and RNA was extracted using the miRNeasy Mini kit (Qiagen) incorporating an on-column RNase-free DNase digestion to remove all DNA.
RNA circularization was performed with CircLigase II ssDNA Ligase. To remove any remaining linear RNA an RNase R digestion was performed. RNA was reverse transcribed using Superscript II and the cDNA was extended with Klenow DNA polymerase I and a tagged primer. The tagged cDNA was amplified using the Failsafe PCR system using primers incorporating Illumina index sequences. PCR products were size selected using Agencourt AMPure XP magnetic beads and library size distribution was analyzed by D1000 ScreenTape analysis and then quantitated by qPCR.

tag: knock out

value: 11 weeks


title: cRNA Mrpp3 rep3

description: mitochondrial RNA


title: cRNA Mrpp3 rep1

description: mitochondrial RNA


title: 221_K562-A


title: 141_K562-B


title: cRNA WT rep2

description: mitochondrial RNA

value: WT


title: 110_K562-B


title: cRNA WT rep1

description: mitochondrial RNA


title: cRNA WT rep3

description: mitochondrial RNA


title: 102_K562-B


title: 79_K562-B


title: 58_K562-B


title: WT standard CircLigase II

description: mitochondrial RNA


title: 215_K562-A


title: 72_K562-B


title: K562_bulkB

extract_protocol:
2-4 million cells were fixed for 10 minutes by resuspending the cell pellet in 5 ml full culture medium supplemented with 1% formaldehyde (Sigma). The reaction was quenched by addition of 2 M glycine to a final concentration of 125 mM and incubation for 5 minutes on ice. Cells were pelleted by centrifugation, washed by resuspension in 1 ml of PBS and pelleted again. Then the cells were resuspended in 1.5 ml of lysis buffer and incubated on ice for at least 15 minutes. Cells were pelleted by centrifugation at 2,500g for 5 minutes, resuspended in 100 μl of 1×NEBuffer 3 and pelleted again. The pellet was resuspended in 100 μl of 0.3% SDS in 1×NEBuffer 3 and incubated at 37°C for 1 hour. Then the suspension was diluted with 330 μl of 1× NEBuffer 3 and 53 μl of 20% Triton X-100 (Sigma), and incubated at 37°C for 1 hour to quench SDS. The cells were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 250 μl of 1× DpnII buffer (NEB). 20 μl were taken as a chromatin integrity control, then 600 U DpnII enzyme (NEB) were added, and the chromatin was digested overnight at 37°C with rotation. In the morning 200 more units of DpnII were added and the cells were incubated for additional 2 hours. DpnII was inactivated by incubation at 65°C for 20 minutes. 20 μl was taken as a digestion control. Nuclei were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 100 μl of 1× T4 DNA ligase buffer (Thermo Scientific) and pelleted again. The pellet was resuspended in 500 μl of 1× T4 DNA ligase buffer, and 50 U T4 DNA ligase (Thermo Scientific) were added. The sample was mixed by inversion and incubated with rotation for 4 hours at 16°C and then for 30 minutes at room temperature. Then the nuclei were pelleted by centrifugation at 3,000g for 5 minutes and resuspended in 120 μl of sterile PBS. 20 μl were taken as a ligation control and another 20 ul were used to for DNA purification, library preparation and sequencing.
The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample  preparation protocol for Solexa platforms (NEB reagents).


title: Mrpp3 rep1

description: mitochondrial RNA

data_processing:
Sequenced reads were trimmed of adapters with cutadapt v1.10 using default parameters and specifying a minimum lengh of 15 nt.
Trimmed reads were aligned with bowtie2 using default parameters to the mouse reference genome sequence (mm10) which had been masked for numts regions, as defined by the UCSC mm9 numtS track with coordinates converted to mm10 with UCSC liftOver.
Alignments were filtered using to remove true multireads (MAPQ >= 2) and coverage was calculated with BEDtools, normalised to total reads mapped to chrM and converted to wig format.
The alignments were futher filtered with bedtools intersect to extract reads that overlap tRNA regions by at least 80%, allowing for 3’ extensions (-s -f 0.8), or reads that overlap rRNA and mRNA regions entirely, excluding true 3’ poly(A) tails (-s -f 1.0). The tRNA-, rRNA- and mRNA-overlapping alignments were filtered with Picard tools using the -CCA and -polyA read ID lists to extract tRNA-aligned reads with 3’ -CCA ends, and rRNA- and mRNA-aligned polyadenylated reads, excluding the true 3’ poly(A) tail.
The coverage of total and 3’ -CCA tRNA-aligned reads at the 3’  terminal nucleotide was computed in each sample and the ratio of the two calculated. The coverage of total and poly(A) rRNA- and mRNA-aligned reads was calculated similarly and converted to wig format.
Genome_build: mm10
Supplementary_files_format_and_content: wig files for each sample representing the normalised coverage for whole read lengths, 5’ and 3’ ends

extract_protocol:
Mitochondria were isolated from homogenized hearts and purified by differential centrifugation and RNA was extracted using the miRNeasy Mini kit (Qiagen) incorporating an on-column RNase-free DNase digestion to remove all DNA.
Libraries were prepared using Illumina TruSeq Small RNA Library methods (adapter: TGGAATTCTCGGGTGCCAAGG). A size selection for RNAs between X and Y was performed.


title: WT rep1

description: mitochondrial RNA


title: 4M M5

status: Public on Mar 22 2017

city: Tallahassee

first: Joseph

middle: Ligon

last: Bundy

state: FL

address: 1115 W Call St

department: Department of Biomedical Sciences

institute: Florida State University

description: sample name: 1056H

data_processing:
Sequences from Illumina adapters were trimmed using Trimmomatic (version 0.32) using the TruSeq3-SE.fa adapter sequence file provided with the program. A minimum length of 50 bases (MINLEN:50) was chosen as a cutoff for each read and 2 seed mismatches were allowed. All reads between 50 and 100 bases were included in further analyses.
The trimmed sequencing reads were aligned to the mouse genome (genome release GRCm38) using Tophat (2.0.13, --read-mismatches and --read-edit-dist set to 3). A General Transfer Format (GTF) file was used with Tophat (--GTF option) for the purpose of gene annotation (Mus_musculus.GRCm38.68.gtf).
Following mapping reads were futher processed (filtered, sorted and indexed) with Samtools and only reads that mapped to a single gene were used for further analysis.
The uniquely mapped reads were used to generate counts for each annotated gene using easyRNASeq (from Bioconductor version 3.0.2, geneModels summarization)
Differential expression analysis of count tables for female and male mice of various ages was performed using the default settings of DESeq2 (1.8.1, Benjamini-Hochberg FDR correction).
Cufflinks (2.1.1, --no-effective-length-correction, --max-bundle-length 10000000 --GTF Mus_musculus.GRCm38.68.gtf) was used to generate FPKM (fragments per kilobase per million reads) normalized values.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Microsoft Excel Worksheet (.xlsx). Contains unnormalized counts generated by easyRNASeq and normalized FPKM values generated by Cufflinks. The raw counts were used by DESeq2 for differential expression analysis.

source_name: Hippocampus

growth_protocol: 2 month old Male 5XFAD mice and female C57BL/6J mice were ordered from The Jackson Laboratory (Bar Harbor, ME). Hemizygous male transgenics and female WT animals were crossed yielding both transgenic and wild-type animals. Animals used in this study were all non-transgenic wild-types. Animals were group housed and kept on a 12 hour light-dark cycle. Food and water were provided ad libitum.

extract_protocol:
The hippocampus (from both sides of the brain) was dissected and rapidly frozen in liquid nitrogen and stored at -80 degrees. Total RNA was extracted from the entire hippocampus from each animal using TRI Reagent (Sigma-Aldrich) according to manufacturer’s directions.
RNA from the dissected hippocampal tissue was extracted using a miRNeasy minikit (Qiagen, catalog #217004) with MaXtract high density columns (Qiagen, catalog #129056). A set of synthetic RNA standards were then added to 5 μg of RNA from each sample as spike-ins to assess the performance of each library (ERCC ExFold RNA Spike-In Mixes Ambion, catalog #4456739). mRNA was purified with a NEBNext mRNA magnetic isolation module (NEB, catalog #E7490L).  cDNA libraries were generated from 50 ng of isolated mRNA using a NEBnext Ultra mRNA library preparation kit for Illumina sequencers (NEB, catalog #E7530L), and a unique 6-nucleotide index was incorporated to each library (NEB, catalog #E7335S, E7500S). The concentration of each cDNA library was estimated with qPCR using Illumina sequencing primers (KAPA Biosystems catalog # KK4835), and average fragment length was determined with a bioanalzyer high sensitivity DNA chip (Agilent Technologies, catalog #5076-4626).  12nM of each cDNA library was pooled into one of three cDNA library pools. Each of the three cDNA pools underwent additional quality control analysis via bioanalyzer and KAPA-PCR. 10pM of each cDNA library pool was sequenced on an Illumina HiSeq 2500

tag: Sex

value: Male

value: 4 Month

value: Hippocampus


title: 156_K562-A


title: 4M M4

description: sample name: 1056G


title: WT 5x CircLigase II

description: mitochondrial RNA


title: 4M M3

description: sample name: 1048K


title: 123_K562-B


title: WT rep2

description: mitochondrial RNA


title: MH-R2

city: Cambridge

first: Anton

last: Goloborodko

state: Massachusetts

address: 77 Massachusetts Ave., E25-524

department: Physics

institute: MIT

data_processing:
Library strategy: Hi-C
Mapping and filtering contacts: We mapped sequenced read pairs to W303 yeast genome using Bowtie 2.1.0 and the previously described method of iterative mapping (Imakaev, M. et al. Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9, 999-1003, doi:10.1038/nmeth.2148 (2012)). To generate contact lists, we assigned each mapped side to a HindIII fragment and removed the contacts with both sides assigned to the same HindIII fragment, reads with one unmapped side as well as PCR duplicates, i.e. identical contact pairs.
Aggregating and normalizing contact maps: To generate Hi-C contact maps, we aggregated filtered contact lists into 10kb genomic bins. We removed the contacts within the first two diagonals of the maps as they are contaminated by uninformative Hi-C artifacts, unligated and self-ligated DNA fragments. Finally, we iteratively corrected the resulting maps to equalize genomic coverage.
Genome_build: w303
Supplementary_files_format_and_content: matrices.tar.gz - files contain sparse tables with Hi-C contact maps at 10kb resolution.

organism: Saccharomyces cerevisiae

source_name: Hi-C, cohesin mutant arrested in metaphase, biological replicate 2

growth_protocol: Yeast cells were grown in YP + 2% Glucose at 25 °C, transferred to YP + 2% Raffinose (YPR) and grown over night to log phase.

extract_protocol:
Hi-C was performed as described in (JM Belton et.al., The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer, Cell Reports, 13(9):1855-67, (2015)) with the variation that cells were fixed at 37 °C.
Hi-C: The yeast culture was crosslinked with 37% formaldehyde to a final concentration of 3% in the culture media for 20 min at 37˚C. The crosslinking was quenched by adding 2X the volume of formaldehyde used of 2.5M Glycine to the culture and incubated culture for 5 min at 25˚C. Cells were lyses by grinding in a cold mortar and pestle. The chromatin was solubilized by adding 1% w/v SDS and Incubating at 65˚C water bath for 10 min. Excess SDS was quenched using 10% v/v Triton X-100. The chromatin was digested with HindIII at 37˚C overnight in a water bath. Digested ends were filled in with biotin-14-dCTP with Klenow at 37˚C for 2 hr in a water bath. The klenow was inactivated by Incubating in a 65˚C water bath for 20 min. Crosslinked and digested fragments were ligated together in a dilute reaction inorder to favor intra-molecular ligation in a 16˚C water bath for 8 hr. Crosslinkes were reversed by incubating the ligation mixture with proteinase K in a 65˚C water bath. Ligation products were purified with phenol (pH 8.0):chloroform and precipitated with sodium acetate pH 5.2 and ethanol. The precipitated DNA was resuspended in 1X TE and then the DNA was re-extracted with phenol (pH 8.0):chloroform and precipitated with sodium acetate pH 5.2 and ethanol. The DNA pellet was dried thoroughly and excess salt was washed out of the solution using an amicon 30kDa spin column using 1X TE. Then the sample was concentrated using a 30kDa amicon column. RNA was degrade using DNase free RNase A at 37˚C for 1 hr. The Hi-C library was quantified on a 0.8% agarose gel in 0.5X TBE by comparing it to known concentration standards. The Hi-C efficiency was accessed by first PCR amplifing a neighboring interaction in the Hi-C library. This amplicon was split and the aliquots were digested with either HindIII, NheI, or HindIII and NheI at 37˚C overnight. The digested products were quantified on a gel and the percent Hi-C efficiency was calculated as the percent digested with NheI divided by the percent digested in the NheI and HindIII combined reaction. Biotin was removed from un-ligated ends using T4 DNA polymerase at 20˚C for 4hrs and then at 75˚C for 20 min to inactivate the enzyme. The DNA was sheared to 50-700bp using the Covaris S2 sonicator. The DNA ends were repaired using T4 DNA Polymerase, T4 Polynucleotide Kinase, and Klenow fragment of DNA Polymerase I at 20˚C for 30 min. The DNA was purified with one Qiagen MinElute Column. A dATP was added to the 3’ end of the molecules by using Klenow Fragment (exo-) at 37˚C for 30 min and then at 65˚C for 20 min to inactivate the enzyme.
Hi-C library construction was performed as described in (JM Belton et.al., The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer, Cell Reports, 13(9):1855-67, (2015)). The end repaired and A-tailed library was fractionated to 100-300bp using 0.9x and then 1.1x ampure XP extractions. For each 1.0 ng of biotinylated ligation products, 1.0 µl of Dynabeads MyOne Strepavidin C1 beads were used to enrich the library. Illumina PE adapters were ligated to the library using NEB Quick Ligase at room temperature for 15 min. The library was amplified using Illumina primers PE1.0 and PE2.0 for as few cycles as possible. Cycle number was determined by a titration experiment that revealed which cycle would produce enough library for sequencing but didn’t produce higher molecular weight artifacts. The library was purified once more with 1.8x Ampure XP to remove primers and primer dimers. The final library was quantified on a bioanalyzer. The libraries were sequenced on either the GAII or HiSeq platforms

treatment_protocol: Cells were arrested in G1 with 10 μg/ml alpha factor until 90% of cells were unbudded (120 min). The cells were washed three times with YPR and released in YPR. After 30 min 10 μg/ml of nocodazole was added and budding was checked after 1h. After cells entered G2 (60-70 min after release), Galactose at 2% final concentration and 15 min later Doxycycline at 50μg/ml final concentration were added. 30 min after addition of Galactose, the temperature was shifted to 37 °C. Cells were grown at 37 °C for 1h. Then cells were washed three times in YPR + Galactose + Doxycycline and released in the same media. Cells were collected after 30 min for the cdc20 metaphase. For cdc45-td  cells, as above except alpha factor arrested samples incubated at 37°C and 2% galactose added before releasing into the cellcycle in the same media at 37°C. Sample were then collected after 100 minutes for Hi-C analysis.

value: cdc20-td + scc1-73 TRP1


title: C-R1

source_name: Hi-C, cdc45-degron arrested in early mitosis with nocodazole, biological replicate 1

value: UBR1::pGAL-myc-UBR1 (HIS3), CDC45::cdc45-td (CUP1p-Ub-DHFRts-HA-CDC45)(TRP1)


title: 4M M2

description: sample name: 1048I


title: K562_bulkA


title: MH-R1

source_name: Hi-C, cohesin mutant arrested in metaphase, biological replicate 1


title: 4M M1

description: sample name: 1048H


title: 4M F5

description: sample name: 1057D

value: Female


title: CH-R1

source_name: Hi-C, cdc45-degron cohesin mutant arrested in early mitosis with nocodazole, biological replicate 1

value: UBR1::pGAL-myc-UBR1 (HIS3), CDC45::cdc45-td (CUP1p-Ub-DHFRts-HA-CDC45)(TRP1), scc1-71, trp1::hphNT1


title: WT3

city: Houston

first: Shao-cong

last: Sun

state: TX

address: 7455 Fannin St.

department: Immunology

institute: MD Anderson Cancer Center

data_processing:
RNA-Seq libraries were prepared and sequencing was performed by the MD Anderson Cancer Center Sequencing and Microarray Facility
The raw reads were aligned to the Mouse reference genome build mm10, using Tophat2 RNASeq alignment software.
HTseq software is used to summarize the gene expression counts from Tophat alignment data after sorting the BAM files.
The raw counts are normalized and differential expression analysis is performed between KO and WT samples using the DEseq2 package.
Genome_build: UCSC mm10
Supplementary_files_format_and_content: Gene-Level Read Count files

source_name: splenic dendritic cells

extract_protocol:
Fresh splenic dendritic cells were isolated from young WT and TBK1-DKO mice (8-week old) and used for total RNA isolation with Trizol (Invitrogen), and subjected to RNA-seq analysis.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: spleen

value: 10-week old

value: wild type

value: dendritic cells (DCs)


title: MD-R2

source_name: Hi-C, condensin mutant arrested in metaphase, biological replicate 2

value: cdc20-td + SMC2 5' upstream -100 to -1 replaced with kanMX-tTA (tetR-VP16)-tetO2  - Ub -DHFRts - 3xHA extended linker), trp1-1::pFA6 TRP1- GAL1-SMC2-K38I-HA-TRP1


title: Mrpp3 rep2

description: mitochondrial RNA


title: M-R2

source_name: Hi-C, WT arrested in metaphase, biological replicate 2

value: MATa ade2-1 ura3-1 his3-11, trp1-1, can1-100 UBR1::pGAL-myc-UBR1 (HIS3), leu2-3 LEU2::pCM244 x3, cdc20-td CDC205' upstream -100 to -1 replaced with kanMX-tTA (tetR-VP16)-tetO2  - Ub -DHFRts - Myc -linker)


title: MD-R1

source_name: Hi-C, condensin mutant arrested in metaphase, biological replicate 1


title: WT2


title: 4M F4

description: sample name: 1066D


title: 54_K562-B

source_name: chronic myelogenous leukemia cells


title: B6 F IMQ 1

source_name: skin

value: female

tag: treatment

value: IMQ


title: M-R1

source_name: Hi-C, WT arrested in metaphase, biological replicate 1


title: 4M F3

description: sample name: 1056E


title: WT1


title: KO3


title: G1-R2

source_name: Hi-C, WT arrested in G1, biological replicate 2


title: 4M F2

description: sample name: 1057B


title: 4M F1

description: sample name: 1057A


title: 2M M5

description: sample name: 1053M

value: 2 Month


title: 2M M4

description: sample name: 1053L


title: 2M M3

description: sample name: 1053K


title: 2M M2

description: sample name: 1052O


title: 2M F5

description: sample name: 1052C


title: Yap-mt rep2

city: SAN FRANCISCO

first: Chuwen

last: Lin

state: California

address: 555 MISSION BAY BLVD S, RM 212A

institute: University of California San Francisco

data_processing:
Tophat for alignment
Cuffdiff for DEG analysis
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include DEG

source_name: lung

extract_protocol:
Extracted by Rneasy Column (Qiagen) following manufacturer's instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: lung

tag: developmental stage

value: Yap-flox/flox; Shh-Cre/+


title: 2M M1

description: sample name: 1050Q

value: Male


title: 2M F4

description: sample name: 1050C

value: Hippocampus


title: 2M F3

description: sample name: 1050B


title: WT rep1


title: Yap-mt rep1


title: preconditioned control 2

city: Edinburgh

first: John

middle: Paterson

last: Thomson

address: Crewe Road

department: MRC Human Genetics Unit

country: United Kingdom

institute: University of Edinburgh

description: preconditioned_control_RNAseq_2 [RD12]

data_processing:
Paired ends aligned using FASTQ paired-end parser
Quality control and Tophat 1.20 Alignment
General sequence statistics calculated
Quantitated Gene expression patterns
Cuffdiff analysis to determine differentially expressed transcripts
Genome_build: mm9
Supplementary_files_format_and_content: .txt file with FPKM expression values carried out using the Cufflinks tool.

source_name: Murine peripheral blood leukocytes (hypoxic preconditioned)

extract_protocol:
Murine peripheral blood leukocytes (1x106/condition) were lysed and RNA extracted using the mirVana™ total RNA isolation protocols (Ambion, Austin, USA). For RNA quantification, samples were treated with DNase (Ambion) and random hexamer cDNA synthesized by reverse transcription. Assays-on-demand gene expression TaqMan® MGB 6FAM dye-labeled products (Applied Biosystems) were used for relative quantification of cDNA.
Illumina Tru-seq paired end strand specific sequencing (Illumina, USA) was carried out on a NextSeq-550 sequencer

tag: strain background

tag: gender

value: Wild type


title: preconditioned infected 2

description: preconditioned_infected_RNAseq_2 [RD09]

tag: genotype/variation

value: bacterial infection


title: preconditioned control 1

description: preconditioned_control_RNAseq_1 [RD10]


title: WT rep2


title: Yap-mt rep3


title: 2M F2

description: sample name: 1053D

value: Female


title: WT rep3


title: 2M F1

description: sample name: 1052I


title: preconditioned infected 1

description: Preconditioned_infected_RNAseq_1 [RD07]


title: G1-R1

source_name: Hi-C, WT arrested in G1, biological replicate 1


title: 1M M5

description: sample name: 1072B

value: 1 Month


title: 1M M4

description: sample name: 1071X


title: 1M M3

description: sample name: 1071T


title: 1M M2

description: sample name: 1071Q


title: Naïve (normoxia) control 1

description: Naïve_control_RNAseq_1 [RD04]

source_name: Murine peripheral blood leukocytes (normoxia)


title: Naïve (normoxia) infected 2

description: Naïve_infected_RNAseq_2 [RD02]


title: 1M M1

description: sample name: 1071P

source_name: Hippocampus


title: Naïve (normoxia) infected 1

description: Naïve_infected_RNAseq_1 [RD01]


title: KO1

value: spleen

tag: cell type


title: 1M F5

description: sample name: 1071I

tag: tissue


title: KO2

tag: age

value: 10-week old


title: 1M F4

description: sample name: 1071H


title: 1M F1

description: sample name: 1071D


title: 1M F2

description: sample name: 1071E


title: Blood Derived Macrophages 3

city: Madison

first: Christian

last: Capitini

state: WI

address: 1111 Highland Ave, WIMR 4137

institute: University of Wisconsin

description:
RNAseq MEM-all the genes.xls
Group1-1671

data_processing:
Trimmomatic version 0.32; adapters:2:20:7:1:true, leading:5, trailing:5, slidingwindow:4:10, minlen:21
Log2 transformed FPKM (fragments per kilobase exon-model per million reads mapped) are used for downstream analyses
Expression quantification is done using Cufflinks24 (version 2.2.0).
Log2 transformed FPKM (fragments per kilobase exon-model per million reads mapped) are used for downstream analyses.
Genome_build: hg19

source_name: Human macrophage

growth_protocol: Macrophages (MØs) were isolated from PBMCs on Day +0. MSCs were cultured in alpha MEM media (Corning CellGro, Manassas, VA) supplemented with 10% heat inactivated FBS (Hyclone, Logan, UT), 100X L-Ala-L-Glutamine (Corning GlutaGro), and 100X NEAA (Corning) and frozen at passage 4 or 5.

extract_protocol:
On day +10, CD14+ cells were collected using Stem Pro Accutase cell detachment (Gibco Life Technologies) and re-sorted based on CD14 to eliminate the MSCs in the MEM cultures.
Libraries were prepared to a mean size of 300-500 base pairs and analyzed using the High Sensitivity DNA Kit (Agilent Technologies) with the Bioanalyzer.  Libraries were then diluted 1:10,000 and analyzed using the Kapa Library Quant Kit (Kapa Biosystems, Wilmington, MA) on the LightCycler96 (Roche Diagnostics, Indianapolis, IN).

treatment_protocol: On day +7, MØs were supplemented with fresh media or incubated with human BM-derived MSCs to obtain MEMs, at a ratio of 10: 1 of MØs:MSCs.

value: Macrophage


title: Blood Derived Macrophages 1

description:
RNAseq MEM-all the genes.xls
Group1-1666


title: Blood Derived Macrophages 2

description:
RNAseq MEM-all the genes.xls
Group1-1669


title: MSC Educated Macrophages 2

description:
RNAseq MEM-all the genes.xls
Group2-1674


title: BM Derived Macrophages 2

description:
RNAseq MEM-all the genes.xls
Group3-1676


title: MSC Educated Macrophages 3

description:
RNAseq MEM-all the genes.xls
Group2-1663


title: 1M F3

description: sample name: 1071G


title: MSC Educated Macrophages 1

description:
RNAseq MEM-all the genes.xls
Group2-1672


title: BM Derived Macrophages 3

description:
RNAseq MEM-all the genes.xls
Group3-1678


title: 404_Sirt1_i73

city: San Diego

first: Maria

middle: Cecilia

last: Marcondes

state: CA

address: 3525 John Hopkins Ct. #200

institute: San Diego Biomedical Research Institute

description: SIV Young experiment 1

data_processing:
Sequence Analysis - The 50-nt and 75-nt sequence reads identified using Illumina’s Hi-Seq and NextSeq, respectively, were mapped to the genome using the BWA algorithm with default settings.
The ChIP DNA was processed into an Illumina ChIP-Seq library and sequenced to generate >2 million reads, which were aligned to the M.mulatta genome annotation (MacaM/Oct 2014 assembly) and 6.2 million unique aligns (removed duplicates) were obtained
A signal map showing fragment densities along the genome was visualized in the Integrated Genome Browser (IGB) and MACS peak finding was used to identify peaks. Control data was derived from 5.1 million (positive control) and 5.8 million (negative control) alignments. With default settings, 2141 Sirt-1 meaningful peaks genome-wide were identified.
Genome_build: MacaM/Oct 2014 assembly
Supplementary_files_format_and_content: Displays the fragment density for 32-nt bins along the genome and is used in peak finding using MACS.

organism: Macaca mulatta

source_name: Microglia

growth_protocol: Brain immune cells were isolated from all representative areas of the brain (free of meninges), by enzymatic digestion of minced tissue, followed by Percoll (Sigma-Aldrich) gradient.

extract_protocol:
Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine.  Lysates were sonicated with a microtip sonicator to shear the DNA. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. For each ChIP reaction, 30 ug of microglia chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reaction was set up using precleared chromatin and Sirt1 antibody (Millipore; lot number 2273971; 4ug per ChIP reaction) and incubated overnight at 4 C. Protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Immune complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on on Illumina’s HiSeq2500 or NextSeq 500 (50 or 75 nt reads, respectively, single end). Reads were aligned to the Rhesus monkey genome (rheMac2) using the BWA algorithm (default settings). Duplicate reads were removed and only uniquely mapped reads (mapping quality >= 25) were used for further analysis. Alignments were extended in silico at their 3’-ends to a length of 200 bp, which is the average genomic fragment length in the size-selected library, and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peak locations were determined using the MACS algorithm (v1.4.2) with a cutoff of p-value = 1e-7.

tag: pathology

value: SIV positive

value: Brain tissue derived microglial cells

value: anti-Sirt-1 (Millipore; lot number 2273971; 4ug per ChIP reaction)


title: 516_Sirt1_i8

description: Uninfected Young experiment 1

value: Uninfected


title: Naïve (normoxia) control 2

description: Naïve_control_RNAseq_2 [RD06]


title: 417_Sirt1_i94

description: SIVE Young experiment 1

value: SIV positive with encephalitis