title: Th17 72h rep2

source_name: Cord blood CD4+ cells 72h after activation and polarization towards Th17


title: C57BL/6 Th17 24h rep2

status: Public on Apr 07 2016

type: SRA

city: Espoo

first: Sini

last: Rautio

address: Konemiehentie 2

country: Finland

institute: Aalto University

description: Th17 polarization was initiated with IL-6 (20 ng/ml, R&D Systems), IL1β (10 ng/ml, R&D Systems) and TGFβ (1 ng/ml, R&D Systems)

library_source: transcriptomic

data_processing:
Illumina Casava1.8 software used for basecalling.
Samples Prof3_1, Prof3_2,Prof3_3, Prof3_4,Prof3_5 were truncated to 50nt and only R1 fastq files were used
Sequence reads were mapped using Tophat (version 1.3.2) with default parameters to the GRCh37 release 63 human reference genome and NCBIM37 release 63 mouse reference genome.
Expression levels were estimated for Ensembl genes using Python script rpkmforgenes with parameters -readcount -no3utr -rmnameoverlap -bamu (Ramsköld et al.,2009). 3' untranslated regions were ignored and regions where several transcripts with different gene identifiers overlap were removed. Genes with RPKM values <3 in at least two replicates at all time points were filtered out from the downstream analysis.
Human lncRNAs were obtained from Gencode (http://www.gencodegenes.org) database and mouse lncRNAs from NONCODE (http://noncode.org). Expression values of lncRNAs were calculated using Python package HTSeq (http://www-huber.embl.de/users/anders/HTSeq), where overlapping reads with protein-coding genes were excluded. Lncrnas with RPKM values <0.5 in at least two replicates at all time points were filtered out from the downstream analysis.
Genome_build: human: hg19
Genome_build: mouse: mm9
Supplementary_files_format_and_content: rc_human_samples.txt: Read count measurements of Ensembl genes
Supplementary_files_format_and_content: rc_mouse_samples.txt: Read count measurements of Ensembl genes
Supplementary_files_format_and_content: rc_human_lncrna.txt: Read count measurements of lncrnas
Supplementary_files_format_and_content: rc_mouse_lncrna.txt: Read count measurements of lncrnas

molecule: total RNA

organism: Mus musculus

source_name: Mouse CD4+ cells 24h after activation and polarization towards Th17

growth_protocol:
human samples: Human CD4+ T cells were purified from umbilical cord blood of healthy neonates (Turku University Central Hospital, Finland). Mononuclear cells were isolated (Ficoll-Paque, Amersham Biosciences) after which CD4+ cells were collected (Dynal CD4 Positive Isolation Kit, Invitrogen). Cells were activated with plate-bound αCD3 (750 ng/24w culture plate well, Immunotech, France) and soluble αCD28 (1 µg/ml, Immunotech) in density of 0.5x106 cells/ml of X-vivo 20 serum free medium (Lonza) supplemented with 2 mM L-Glutamine (#G7513, Sigma), 50 U/ml Penicillin and 50 μg/ml Streptomycin (#P0781, Sigma) and neutralizing αIFNg (1 µg/ml) and αIL4 (1 µg/ml), and cultured at 37°C in 5 % CO2.
mouse samples: Naïve CD4 T cells (CD4+CD25-CD44lo) were isolated from C57BL/6 mice and the cells were sort-purified and cultured in IMDM (Sigma-Aldrich) supplemented with 5% FCS, 2x10-3 M L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 5x10-5 M β-mercaptoethanol (all Sigma) in the presence of plate-bound α-CD3 (0.5 µg/ml; 2C11) and plate-bound α-CD28 (5 µg/ml; 37.51; both from Large Scale Facility, Medical Research Council National Institute for Medical Research).

extract_protocol:
RNA from human and mouse samples was extracted with RNeasy mini kit (Qiagen) according to manufacturer's instructions. In-column DNase (RNase-Free DNase Set, Qiagen) treatment was performed for 15 minutes. RNA was eluted in dH2O.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: source

tag: cell type

tag: mouse biological replicate


title: Th17 6h rep2

description: Th17 polarization was initiated with IL-6 (20 ng/ml, Roche), IL1β (10 ng/ml, R&D Systems) and TGFβ (10 ng/ml, R&D Systems)

organism: Homo sapiens

source_name: Cord blood CD4+ cells 6h after activation and polarization towards Th17

value: Cord blood

tag: human biological replicate


title: Th17 48h rep2

source_name: Cord blood CD4+ cells 48h after activation and polarization towards Th17


title: Th17 12h rep2

source_name: Cord blood CD4+ cells 12h after activation and polarization towards Th17


title: C57BL/6 Th17 12h rep2

source_name: Mouse CD4+ cells 12h after activation and polarization towards Th17


title: Th17 2h rep2

source_name: Cord blood CD4+ cells 2h after activation and polarization towards Th17


title: Th17 4h rep2

source_name: Cord blood CD4+ cells 4h after activation and polarization towards Th17


title: C57BL/6 Th17 0.5h rep2

source_name: Mouse CD4+ cells 0.5h after activation and polarization towards Th17


title: C57BL/6 Th17 2h rep2

source_name: Mouse CD4+ cells 2h after activation and polarization towards Th17


title: C57BL/6 Th17 4h rep2

source_name: Mouse CD4+ cells 4h after activation and polarization towards Th17


title: C57BL/6 Th17 24h rep1


title: Th17 1h rep2

source_name: Cord blood CD4+ cells 1h after activation and polarization towards Th17


title: C57BL/6 Th17 1h rep2

source_name: Mouse CD4+ cells 1h after activation and polarization towards Th17


title: C57BL/6 Th17 72h rep1

source_name: Mouse CD4+ cells 72h after activation and polarization towards Th17


title: Th17 0.5h rep2

source_name: Cord blood CD4+ cells 0.5h after activation and polarization towards Th17


title: C57BL/6 Th17 6h rep1

source_name: Mouse CD4+ cells 6h after activation and polarization towards Th17


title: parental_H3K4me1_ChIP_rep1

city: Shanghai

first: Yang

last: Shi

address: NO. 138, Yi-Xue-Yuan Road

country: China

institute: Fudan university

library_source: genomic

data_processing:
base_calling: CASAVA package (Illumina v1.7)
alignment: bowtie (version 1.0.0)
peak_calling: macs14 (version 1.4)
Genome_build: hg19

molecule: genomic DNA

source_name: ZR-75-30 cell

growth_protocol: ZR-75-30 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco).

extract_protocol:
Chromatin samples were incubated with specific antibodies in ChIP Lysis buffer (50mM HEPES pH7.5, 500mM NaCl, 1mM EDTA, 1%Triton, 0.1%Na-deoxylcholate, 0.1% SDS) overnight at 4℃. The protein-DNA complexes were immobilized on protein A/G beads (10μl per reaction). The bound fractions were washed 3 times with Lysis buffer, and 3 times with RIPA buffer (50mM Hepes, 300mM LiCl, 1mM EDTA, 0.5%NP-40, 0.7%Nadeoxylcholate), and once with TE. Elution and Reverse crosslinking were carried out in Elution buffer (50mM Tris, PH8.0, 1mM EDTA, 1% SDS) 65℃ for 6 hours. After RNase A and protease K digestion, DNA fraction was purified using PCR extraction kit (QIAGEN).
ChIP DNA libraries were prepared for sequencing using standard Illumina protocols.

tag: antibody

value: ActiveMotif, 39297

tag: cell line

tag: genotype

value: parental


title: RACK7_ChIP_rep1

value: Bethyl, A302-090A


title: parental_KDM5C_ChIP_rep1

value: Bethyl, A301-034A


title: KO_H3K4me1_ChIP_rep1

value: RACK7 knockout


title: Th0 6h rep2

source_name: Cord blood CD4+ cells 6h after activation


title: Th0 1h rep2

source_name: Cord blood CD4+ cells 1h after activation


title: C57BL/6 Th17 48h rep1

source_name: Mouse CD4+ cells 48h after activation and polarization towards Th17


title: C57BL/6 Th0 24h rep3

source_name: Mouse CD4+ cells 24h after activation


title: Th0 48h rep2

source_name: Cord blood CD4+ cells 48h after activation


title: Th0 24h rep2

source_name: Cord blood CD4+ cells 24h after activation


title: C57BL/6 Th17 12h rep1

source_name: Mouse CD4+ cells 12h after activation and polarization towards Th17


title: C57BL/6 Th17 4h rep1


title: Th0 72h rep2

source_name: Cord blood CD4+ cells 72h after activation


title: Th0 4h rep2

source_name: Cord blood CD4+ cells 4h after activation


title: C57BL/6 Th17 1h rep1


title: C57BL/6 Th0 6h rep3

source_name: Mouse CD4+ cells 6h after activation


title: Th0 12h rep2

source_name: Cord blood CD4+ cells 12h after activation


title: C57BL/6 Th0 48h rep3

source_name: Mouse CD4+ cells 48h after activation


title: C57BL/6 Th0 4h rep3

source_name: Mouse CD4+ cells 4h after activation


title: C57BL/6 Th0 72h rep3

source_name: Mouse CD4+ cells 72h after activation


title: Th0 0.5h rep2

source_name: Cord blood CD4+ cells 0.5h after activation


title: Th17 24h rep1

source_name: Cord blood CD4+ cells 24h after activation and polarization towards Th17


title: Th17 12h rep1

source_name: Cord blood CD4+ cells 12h after activation and polarization towards Th17


title: Th0 2h rep3

source_name: Cord blood CD4+ cells 2h after activation


title: Th0 2h rep2


title: Thp rep2

source_name: Cord blood CD4+ cells without any treatments


title: C57BL/6 Th0 1h rep3

source_name: Mouse CD4+ cells 1h after activation


title: C57BL/6 Th0 0.5h rep3

source_name: Mouse CD4+ cells 0.5h after activation


title: Th17 2h rep1

source_name: Cord blood CD4+ cells 2h after activation and polarization towards Th17


title: C57BL/6 Th17 0.5h rep1

source_name: Mouse CD4+ cells 0.5h after activation and polarization towards Th17


title: C57BL/6 Th0 12h rep3

source_name: Mouse CD4+ cells 12h after activation


title: C57BL/6 Th17 2h rep1

source_name: Mouse CD4+ cells 2h after activation and polarization towards Th17


title: Thymidine_Chase_rep_2_pd_(2013-12-20.2,2014-01-07.2)

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

country: USA

institute: Fred Hutchinson Cancer Research Center

description: EdU-Biotin pull-down - MINCE-seq with 10 min EdU labeling and 1 hour thymidine chase

data_processing: 1. We used Novoalign (2.08.01 - 17 Oct 2012) to map paired-end 25bp reads to release r5.51 (May 2013) of the D.melanogaster genomic sequence obtained from FlyBase. If a read was mapped to multiple locations, one location was picked at random 2. We extracted properly paired reads (Supplementary file .bed) 3. We selected aligned paired-end fragments by insert size length (134-160 bp for nucleosomal size). 4. For every ten base pairs in the genome, we counted the number of paired-end nucleosomal fragments aligned over that region. 5. We normalized counts over ten-base-pair window by dividing by the total number of counts for all base pairs and then multiplying by the total number of base pairs in the genome (Supplementary file .wig)

organism: Drosophila melanogaster

source_name: Biotin Pulldown

extract_protocol: Cells were grown to saturation and split into fresh media at density of 2x106 cells/ml 20 hours before starting the MINCE protocol (Rizzino and Blumenthal, In Vitro, 1978). Cells were labeled with 10 _M EdU for 10 minutes and rapidly harvested and washed with ice cold PBS. For a thymidine chase, cells were spun down and resuspended in fresh media with 20 _M thymidine for 1 hour. The washed cells were crosslinked with 1% formaldehyde in PBS at room temperature for 15 minutes, quenched with 250 mM glycine for 5 minutes and washed three times with PBS. The cells were then permeabilized with 0.25% Triton X-100 in PBS for 30 minutes at room temperature. Cells were then washed once with 0.5% BSA in PBS. Cells were then resuspended in PBS and click reagents were added in the following order with mixing after each addition: biotin TEG-azide (final concentration: 100 _M), sodium ascorbate (final concentration: 10 mM) and CuSO4 (final concentration: 2 mM). The click reaction mixture was incubated for 1 hour at room temperature. After the reaction, cells were washed three times with PBS, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1), and incubated on ice for 10 minutes. Dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1, 3 mM CaCl2) at 10 times the volume of lysis buffer was added and mixed well. The lysed cell suspension was sonicated with the Branson digital sonifier with following settings: total on time: 40s; power: 30%; pulse: 2.5s on; 5s off. The lysate was incubated at 37°C for 5 minutes, and then MNase was added to the lysate and incubated for 10 minutes. MNase reactions were stopped by addition of EGTA to a final concentration of 2 mM, of SDS to a final concentration of 0.9%, and of proteinase K. The lysate was then incubated at 65°C for at least 12 hours to reverse cross-links and degrade proteins. After incubation, phenol-chloroform-isoamyl alcohol (PCI) extraction was performed and RNAse-A was added to the aqueous phase and incubated at 37°C for 30 minutes. PCI extraction was performed again, and the DNA in the aqueous phase was precipitated using 70% isopropanol, 30 mM sodium acetate and glycoblue at -80°C for at least 30 minutes followed by centrifugation at 4°C, 16,000 rcf for at least 30 minutes. The DNA pellet was washed with 75% ethanol and resuspended in 0.1x TE. After reserving an aliquot for input, biotinylated DNA was pulled down using M-280 streptavidin dynabeads (Life Technologies), by following the manufacturer's protocol. After washes, the beads were resuspended in 0.1X TE followed by addition of SDS to a final concentration of 1% and addition of proteinase K. The beads were incubated at 65°C for 15 minutes followed by PCI extraction and DNA precipitation as above. Input and pull-down DNA were used in a modified Illumina library preparation protocol that captures all fragments between 25 and 500 bp in size (Henikoff et al., Proceedings of the National Academy of Sciences of the United States of America, 2011).


title: Th17 72h rep1

source_name: Cord blood CD4+ cells 72h after activation and polarization towards Th17


title: C57BL/6 Th0 2h rep3

source_name: Mouse CD4+ cells 2h after activation


title: C57BL/6 Th0 72h rep2


title: Th17 4h rep1

source_name: Cord blood CD4+ cells 4h after activation and polarization towards Th17


title: Th17 0.5h rep1

source_name: Cord blood CD4+ cells 0.5h after activation and polarization towards Th17


title: Th17 1h rep1

source_name: Cord blood CD4+ cells 1h after activation and polarization towards Th17


title: C57BL/6 Th0 48h rep2

source_name: Mouse CD4+ cells 48h after activation


title: Thymidine_Chase_rep_1_pd_(2013-12-20.1,2014-01-07.1)


title: Th17 48h rep1

source_name: Cord blood CD4+ cells 48h after activation and polarization towards Th17


title: Th0 72h rep1

source_name: Cord blood CD4+ cells 72h after activation


title: C57BL/6 Thp 0h rep3

source_name: Mouse CD4+ cells without any treatments


title: C57BL/6 Th0 24h rep2

source_name: Mouse CD4+ cells 24h after activation


title: Th0 48h rep1

source_name: Cord blood CD4+ cells 48h after activation


title: Thymidine_Chase_rep_2_input_(2013-12-20.15,2014-01-07.15)

description: Input for MINCE-seq with 10 min EdU labeling and 1 hour thymidine chase

source_name: Input


title: MNase_4X_pd_(2015-11-18.19)

description: EdU-Biotin pull-down - MINCE-seq with 10 min EdU labeling with 4X MNase concentration

extract_protocol: Cells were labeled with 10 _M EdU for 10 minutes and rapidly harvested and washed with ice cold PBS. The washed cells were crosslinked with 1% formaldehyde in PBS at room temperature for 15 minutes, quenched with 250 mM glycine for 5 minutes and washed three times with PBS. The cells were then permeabilized with 0.25% Triton X-100 in PBS for 30 minutes at room temperature. Cells were then washed once with 0.5% BSA in PBS. Cells were then resuspended in PBS and click reagents were added in the following order with mixing after each addition: biotin TEG-azide (final concentration: 100 _M), sodium ascorbate (final concentration: 10 mM) and CuSO4 (final concentration: 2 mM). The click reaction mixture was incubated for 1 hour at room temperature. After the reaction, cells were washed three times with PBS, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1), and incubated on ice for 10 minutes. Dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1, 3 mM CaCl2) at 10 times the volume of lysis buffer was added and mixed well. The lysed cell suspension was sonicated with the Branson digital sonifier with following settings: total on time: 40s; power: 30%; pulse: 2.5s on; 5s off. The lysate was incubated at 37°C for 5 minutes, and then MNase was added to the lysate and incubated for 10 minutes. MNase reactions were stopped by addition of EGTA to a final concentration of 2 mM, of SDS to a final concentration of 0.9%, and of proteinase K. The lysate was then incubated at 65°C for at least 12 hours to reverse cross-links and degrade proteins. After incubation, phenol-chloroform-isoamyl alcohol (PCI) extraction was performed and RNAse-A was added to the aqueous phase and incubated at 37°C for 30 minutes. PCI extraction was performed again, and the DNA in the aqueous phase was precipitated using 70% isopropanol, 30 mM sodium acetate and glycoblue at -80°C for at least 30 minutes followed by centrifugation at 4°C, 16,000 rcf for at least 30 minutes. The DNA pellet was washed with 75% ethanol and resuspended in 0.1x TE. After reserving an aliquot for input, biotinylated DNA was pulled down using M-280 streptavidin dynabeads (Life Technologies), by following the manufacturer's protocol. After washes, the beads were resuspended in 0.1X TE followed by addition of SDS to a final concentration of 1% and addition of proteinase K. The beads were incubated at 65°C for 15 minutes followed by PCI extraction and DNA precipitation as above. Input and pull-down DNA were used in a modified Illumina library preparation protocol that captures all fragments between 25 and 500 bp in size (Henikoff et al., Proceedings of the National Academy of Sciences of the United States of America, 2011).


title: Thymidine_Chase_rep_1_input_(2013-12-20.14,2014-01-07.14)


title: C57BL/6 Th0 12h rep2


title: MNase_1X_pd_(2015-11-18.11)

description: EdU-Biotin pull-down - MINCE-seq with 10 min EdU labeling with 1X MNase concentration


title: Th0 6h rep1

source_name: Cord blood CD4+ cells 6h after activation


title: C57BL/6 Th0 6h rep2

source_name: Mouse CD4+ cells 6h after activation


title: Th0 12h rep1

source_name: Cord blood CD4+ cells 12h after activation


title: MNase_0.5X_pd_(2015-11-18.9)

description: EdU-Biotin pull-down - MINCE-seq with 10 min EdU labeling with 0.5X MNase concentration


title: MNase_4X_input_(2015-11-18.12)

description: Input for MINCE-seq with 10 min EdU labeling with 4X MNase concentration


title: C57BL/6 Th0 4h rep2

source_name: Mouse CD4+ cells 4h after activation


title: Th0 2h rep1

source_name: Cord blood CD4+ cells 2h after activation


title: MNase_0.5X_input_(2015-11-18.8)

description: Input for MINCE-seq with 10 min EdU labeling with 0.5X MNase concentration


title: C57BL/6 Th0 0.5h rep2

source_name: Mouse CD4+ cells 0.5h after activation


title: Th0 1h rep1

source_name: Cord blood CD4+ cells 1h after activation


title: C57BL/6 Th0 2h rep2

source_name: Mouse CD4+ cells 2h after activation


title: Th0 4h rep1

source_name: Cord blood CD4+ cells 4h after activation


title: Long_term_labeling_pd_(2015-11-18.7)

description: EdU-Biotin pull-down - MINCE-seq with 24 hours EdU labeling

source_name: Biotin Pulldown

extract_protocol: Cells were labeled with 10 _M EdU for 24 hours and rapidly harvested and washed with ice cold PBS. The washed cells were crosslinked with 1% formaldehyde in PBS at room temperature for 15 minutes, quenched with 250 mM glycine for 5 minutes and washed three times with PBS. The cells were then permeabilized with 0.25% Triton X-100 in PBS for 30 minutes at room temperature. Cells were then washed once with 0.5% BSA in PBS. Cells were then resuspended in PBS and click reagents were added in the following order with mixing after each addition: biotin TEG-azide (final concentration: 100 _M), sodium ascorbate (final concentration: 10 mM) and CuSO4 (final concentration: 2 mM). The click reaction mixture was incubated for 1 hour at room temperature. After the reaction, cells were washed three times with PBS, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1), and incubated on ice for 10 minutes. Dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1, 3 mM CaCl2) at 10 times the volume of lysis buffer was added and mixed well. The lysed cell suspension was sonicated with the Branson digital sonifier with following settings: total on time: 40s; power: 30%; pulse: 2.5s on; 5s off. The lysate was incubated at 37°C for 5 minutes, and then MNase was added to the lysate and incubated for 10 minutes. MNase reactions were stopped by addition of EGTA to a final concentration of 2 mM, of SDS to a final concentration of 0.9%, and of proteinase K. The lysate was then incubated at 65°C for at least 12 hours to reverse cross-links and degrade proteins. After incubation, phenol-chloroform-isoamyl alcohol (PCI) extraction was performed and RNAse-A was added to the aqueous phase and incubated at 37°C for 30 minutes. PCI extraction was performed again, and the DNA in the aqueous phase was precipitated using 70% isopropanol, 30 mM sodium acetate and glycoblue at -80°C for at least 30 minutes followed by centrifugation at 4°C, 16,000 rcf for at least 30 minutes. The DNA pellet was washed with 75% ethanol and resuspended in 0.1x TE. After reserving an aliquot for input, biotinylated DNA was pulled down using M-280 streptavidin dynabeads (Life Technologies), by following the manufacturer's protocol. After washes, the beads were resuspended in 0.1X TE followed by addition of SDS to a final concentration of 1% and addition of proteinase K. The beads were incubated at 65°C for 15 minutes followed by PCI extraction and DNA precipitation as above. Input and pull-down DNA were used in a modified Illumina library preparation protocol that captures all fragments between 25 and 500 bp in size (Henikoff et al., Proceedings of the National Academy of Sciences of the United States of America, 2011).


title: Long_term_labeling_input_(2015-11-19.6)

description: Input for MINCE-seq with 24 hours EdU labeling


title: Thp rep1

source_name: Cord blood CD4+ cells without any treatments


title: C57BL/6 Th0 1h rep2

source_name: Mouse CD4+ cells 1h after activation


title: EdU_labeling_rep_2_pd_(2013-12-20.13,2014-01-07.13)

description: EdU-Biotin pull-down - MINCE-seq with 10 min EdU labeling


title: EdU_labeling_rep_2_input_(2013-12-20.11,2014-01-07.11)

description: Input for MINCE-seq with 10 min EdU labeling


title: Th0 24h rep1

source_name: Cord blood CD4+ cells 24h after activation


title: MNase_1X_input_(2015-11-18.10)

description: Input for MINCE-seq with 10 min EdU labeling with 1X MNase concentration


title: EdU_labeling_rep_1_pd_(2013-12-20.12,2014-01-07.12)


title: Th0 0.5h rep1

source_name: Cord blood CD4+ cells 0.5h after activation


title: C57BL/6 Thp 0h rep2

source_name: Mouse CD4+ cells without any treatments


title: C57BL/6 Th0 72h rep1

source_name: Mouse CD4+ cells 72h after activation


title: C57BL/6 Thp 0h rep1


title: C57BL/6 Th0 1h rep1


title: C57BL/6 Th0 48h rep1

source_name: Mouse CD4+ cells 48h after activation


title: C57BL/6 Th0 12h rep1

source_name: Mouse CD4+ cells 12h after activation


title: BG3_rep2_input_(2015-01-13.3,2015-03-20.3)

source_name: Input


title: coIP CJ 81-176 csrA-3xFLAG

city: Cologne

first: Konrad

middle: U.

last: Förstner

state: North Rhine-Westphalia

address: Gleueler Str. 60

department: Information Services

country: Germany

institute: ZB MED - Information Centre for Life Sciences

data_processing:
Demultiplexing
Fastq quality trimming using FastX and a cut-off value of 20
Fastq to fasta conversion using FastX
Size filtering: discarding reads shorter than 12 nt (READemption)
Read mapping using segemehl version 0.1.7 (READemption)
Coverage calculation and normalization (READemption)
Genome_build: C. jejuni NCTC11168: ASM908v1 (NC_002163.1); C. jejuni 81-176: ASM1552v1 (NC_008770.1, NC_008787.1, NC_008790.1)
Supplementary_files_format_and_content: wiggle

organism: Campylobacter jejuni subsp. jejuni 81-176

source_name: Campylobacter jejuni 81-176 cells

growth_protocol: C. jejuni wildtype and csrA-3XFLAG strains were grown to mid-exponential phase (~0.6 OD600) in Brucella Broth. Cells corresponding to 60 OD600 were harvested to perform the coIP.

extract_protocol:
Phenol/Chloroform/Isoamyl alcohol RNA extraction (Rieder et. al., RNA Biology 2012)
The libraries were generated by Vertis Biotechnologie AG (Munich, Germany). The samples were poly(A)-tailed by using poly(A) polymerase. The 5'-PPP were removed using tobacco acid pyrophosphatase (TAP) followed by the ligation of the RNA adapter to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed with an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to reach a concentration of 20-30 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina.

treatment_protocol: Cells were lysed and anti-FLAG antibody/sepharose beads were used to co-immunoprecipitate CsrA-3xFLAG and bound RNA.

tag: genotype/variation

tag: rip antibody

value: Antibody-Monoclonal ANTI-FLAG M2 (Sigma, catalog# F1804, Batch no.(lot)# SLBD6976)


title: coIP CJ 81-176 WT

value: WT


title: C57BL/6 Th0 24h rep1

source_name: Mouse CD4+ cells 24h after activation


title: BG3_rep1_input_(2015-01-13.1,2015-03-20.1)


title: mES_Rack7_ChIP

data_processing:
base_calling: CASAVA package (Illumina v1.7)
alignment: bowtie (version 1.0.0)
peak_calling: macs14 (version 1.4)
Genome_build: mES_Rack7_ChIP: mm9, others: hg19

source_name: mES cell

growth_protocol: ZR-75-30 and MCF-7were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco). mES cells were grown on 0.1% gelatinized (Sigma, G1890) tissue culture plates in ESC media. ESC media: DMEM supplemented with 10% fetal bovine serum, 1000 U/mL LIF, 100 µM nonessential amino acids (Invitrogen, 11140-050), 2 mM L-glutamine (Invitrogen, 25030-081), 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen, 15140-122), and 1 μL/mL of 2-mercaptoethanol (Sigma, M7522).


title: EdU_labeling_rep_1_input_(2013-12-20.10,2014-01-07.10)


title: Th17 6h rep1

source_name: Cord blood CD4+ cells 6h after activation and polarization towards Th17


title: BIRD1_gluc_but1

status: Public on Apr 06 2016

city: Malaga

first: Carlos

middle: Molina

last: Santiago

state: Malaga

address: Bulevar Louis Pasteur 49

department: MIcrobiology

country: Spain

institute: UMA

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to BIRD1 whole genome using Rockhopper
Expressions were calculated using Rockhopper
The wig file was obtained using Rockhopper software and contains the position of the differential expressed genes in all the conditions tested.
Genome_build: ASM18364v1
Supplementary_files_format_and_content: .xlsx files include expression values (normalized abundance measurements) for each Sample
Supplementary_files_format_and_content: .wig file includes the position of the differential expressed genes in all the conditions tested.

organism: Pseudomonas putida BIRD-1

source_name: P. putida BIRD1

extract_protocol:
Cells were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: treatment

value: glucose and butanol


title: C57BL/6 Th0 4h rep1

source_name: Mouse CD4+ cells 4h after activation


title: BIRD1_gluc_but2


title: dsGFP_pd_(2015-05-13.9,2015-05-21.9)

description: EdU-Biotin pull-down - MINCE-seq with 10 min EdU labeling from cells treated with control dsRNA

extract_protocol: dsRNA added to the final concentration of 10 _g/ 1x106 cells in serum-free media, mixed well and incubated for 30 minutes at room temperature, followed by supplementation with equal volume of fresh media and FBS to a final concentration of 10%. After 4 days of dsRNA treatment, cells were treated with 10 _M EdU for 10 minutes and rapidly harvested and washed with ice cold PBS. For a thymidine chase, cells were spun down and resuspended in fresh media with 20 _M thymidine for 1 hour. The washed cells were crosslinked with 1% formaldehyde in PBS at room temperature for 15 minutes, quenched with 250 mM glycine for 5 minutes and washed three times with PBS. The cells were then permeabilized with 0.25% Triton X-100 in PBS for 30 minutes at room temperature. Cells were then washed once with 0.5% BSA in PBS. Cells were then resuspended in PBS and click reagents were added in the following order with mixing after each addition: biotin TEG-azide (final concentration: 100 _M), sodium ascorbate (final concentration: 10 mM) and CuSO4 (final concentration: 2 mM). The click reaction mixture was incubated for 1 hour at room temperature. After the reaction, cells were washed three times with PBS, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1), and incubated on ice for 10 minutes. Dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1, 3 mM CaCl2) at 10 times the volume of lysis buffer was added and mixed well. The lysed cell suspension was sonicated with the Branson digital sonifier with following settings: total on time: 40s; power: 30%; pulse: 2.5s on; 5s off. The lysate was incubated at 37°C for 5 minutes, and then MNase was added to the lysate and incubated for 10 minutes. MNase reactions were stopped by addition of EGTA to a final concentration of 2 mM, of SDS to a final concentration of 0.9%, and of proteinase K. The lysate was then incubated at 65°C for at least 12 hours to reverse cross-links and degrade proteins. After incubation, phenol-chloroform-isoamyl alcohol (PCI) extraction was performed and RNAse-A was added to the aqueous phase and incubated at 37°C for 30 minutes. PCI extraction was performed again, and the DNA in the aqueous phase was precipitated using 70% isopropanol, 30 mM sodium acetate and glycoblue at -80°C for at least 30 minutes followed by centrifugation at 4°C, 16,000 rcf for at least 30 minutes. The DNA pellet was washed with 75% ethanol and resuspended in 0.1x TE. After reserving an aliquot for input, biotinylated DNA was pulled down using M-280 streptavidin dynabeads (Life Technologies), by following the manufacturer's protocol. After washes, the beads were resuspended in 0.1X TE followed by addition of SDS to a final concentration of 1% and addition of proteinase K. The beads were incubated at 65°C for 15 minutes followed by PCI extraction and DNA precipitation as above. Input and pull-down DNA were used in a modified Illumina library preparation protocol that captures all fragments between 25 and 500 bp in size (Henikoff et al., Proceedings of the National Academy of Sciences of the United States of America, 2011).


title: BIRD1_shock1

value: butanol shock


title: C57BL/6 Th0 2h rep1

source_name: Mouse CD4+ cells 2h after activation


title: C57BL/6 Th0 0.5h rep1

source_name: Mouse CD4+ cells 0.5h after activation


title: BIRD1_But1

value: butanol


title: dsCaf1_input_(2015-05-13.6,2015-05-21.6)

description: Input for MINCE-seq with 10 min EdU labeling from cells treated with dsRNA to knock-down Caf1-P105


title: BG3_rep1_pd_(2015-01-13.2)

source_name: Biotin Pulldown


title: dsGFP_input_(2015-05-13.5,2015-05-21.5)

description: Input for MINCE-seq with 10 min EdU labeling from cells treated with control dsRNA


title: BG3_rep2_pd_(2015-01-13.4)


title: BIRD1_gluc1

value: glucose


title: BIRD1_shock2


title: FPR13I

city: Brisbane

first: Shuhong

last: Gao

state: QLD

address: Research Road, St Lucia

department: Advanced Water Management Centre

country: Australia

institute: The University of Queensland

data_processing:
NGS QC Toolkit (v2.3.3) was used to do the quality control and tag removal to generate the clean sequence reads; the resulting clean reads no shorter than 75 bp were used for downstream analyses.
The cleaned sequence reads for each sample were aligned to the PAO1 reference genome (NC_002516) using SeqAlto
Strand-specific coverage for each gene was calculated and differential expression analysis was conducted using the cuffdiff command in Cufflinks (version 2.2.1) on triplicated cultures
Statistic analyses and visualization were conducted using the cummeRbund package in R (http://compbio.mit.edu/cummeRbund/). Gene expression was calculated as reads per kilobase of a gene per million mapped reads (RPKM), a normalized value derived from the frequency of detection and the length of a given gene (Cole Trapnell, Nature Protocols, 2012) Differences in fold-change values were calculated between control and FNA-treated samples (0.1 mg N/L) by determining the log2 fold-change (LFC) of the averaged RPKM values determined from triplicate experiments run on two separate occasions.
Genome_build: NC_002516
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

organism: Pseudomonas aeruginosa

source_name: Bacteria

growth_protocol: PAO1 was activated according to supplier's instructions and grown on tryptic soy agar plates at 30 ºC for 26 hours. One colony was then transferred to tryptic soy broth medium and incubated shaken at 150 rpm and 30 ºC for another 26 hours. Afterwards, 5 mL of the bacterium suspension was transferred into serum bottles containing 150 mL of sterile anaerobic GLYM9 medium in an anaerobic chamber. After 26 hours’ incubation the optical density at 600 nm (OD600) of the culture was adjusted to 0.5. Then 10 mL of this culture was transferred into 150 mL of sterile anaerobic GLYM9 medium in serum bottles in an anaerobic chamber and then incubated at 30 ˚C while shaken at 150 rpm.

extract_protocol:
5 mL of the bacteria suspension from each serum bottle was centrifuged at 13,000 x g for 2 minutes, the supernatant was discarded and the pellets were immediately frozen in the liquid nitrogen before storing at -80ºC for later RNA extraction. Total RNA extraction was performed using the QIAGEN miRNeasy Mini Kit (Catalog number: 217004) according to the manufacturer’s instructions except adding an extra bead-beating step to ensure the complete lysis of the bacterial cells.
Strand-specific cDNA library construction

treatment_protocol: When the cultures were in early log phase of growth, eleven hours after inoculation, FNA was added with starting concentration of 0.1 mg N/L. Control cultures (no added FNA) had the same volume of sterilized Milli-Q water added. Experiments of the control and FNA treated cultures were conducted in triplicates. Samples were taken for RNA extraction from the triplicate FNA treated and the triplicate control cultures of PAO1 after 13 hours incubation, that is 2 hours after FNA addition.

tag: strain

value: wild type

tag: incubation time

value: 13 hours incubation after the standard inoculation, that is 2 hours after FNA addition

tag: fna level

value: 0.1 mg N/L


title: BIRD1_gluc2


title: dsCaf1_pd_(2015-05-13.10,2015-05-21.10)

description: EdU-Biotin pull-down - MINCE-seq with 10 min EdU labeling from cells treated with dsRNA to knock-down Caf1-P105


title: C57BL/6 Th0 6h rep1

source_name: Mouse CD4+ cells 6h after activation


title: FPR13H


title: FPR13G


title: FPR13C

value: 13 hours incubation after the standard inoculation


title: FPR13B


title: TY2205 Young Adult Rep3 RNAseq

city: New York

first: Sevinc

last: Ercan

state: NY

address: 12 Waverly Place

department: Biology

institute: New York University

data_processing:
Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated using cufflinks version 2.2.0 using default parameters supplying gene annotations.
Genome_build: WS220
Supplementary_files_format_and_content: FPKM text file contains the FPKM values for each gene for each replicate.

organism: Caenorhabditis elegans

growth_protocol:
Young adults were synchronized by overnight embryo hatching and grown for 44 hours at 25C
Larvae were collected after overnight embryo hatching and growth for 24 hours (L3 larvae) on NGM plates

extract_protocol:
RNA was extracted by freeze cracking embryos by freezein in liquid nitrogen and thawing at 37C water bath five times. RNA was isolated according to the manufacturer's (Invitrogen) Trizol extraction protocol. Purified total RNA RNA was cleaned up through Qiagen RNeasy column.
mRNA was purified from .5-10 ug of total RNA using Sera-Mag Oligo(dT) beads (ThermoScientific). cDNA preparation was done in the presence of dUTP to prepare stranded RNA-seq libraries as in PMID:19620258

treatment_protocol:
Packed adults were resuspended in 10V of Trizol and frozen at -80C.
Worms were resuspended in 10V of Trizol and frozen at -80C.

tag: tissue

value: whole worms

tag: developmental stage

value: young adult

value: mRNA was purified from .5-10 ug of total RNA using Sera-Mag Oligo(dT) beads (ThermoScientific). cDNA preparation was done in the presence of dUTP to prepare stranded RNA-seq libraries as in PMID:19620234


title: CB5362 Young Adult Rep1 RNAseq


title: CB5362 Young Adult Rep2 RNAseq


title: CB5362 Young Adult Rep3 RNAseq


title: TY2205 Young Adult Rep2 RNAseq


title: TY2205 Young Adult RNAseq (REVERTED MUTANT, DATA NOT USED)


title: CB5362 Young Adult Rep4 RNAseq


title: TY2205 Young Adult Rep1 RNAseq


title: TY2205 L3 Rep3 RNAseq

growth_protocol:
Larvae were collected after overnight embryo hatching and growth for 24 hours (L3 larvae) on NGM plates
Larvae were collected after overnight embryo hatching and growth for 24 hours (L3 larvae) on NGM plates

treatment_protocol:
Packed larvae were resuspended in 10V of Trizol and frozen at -80C.
Worms were resuspended in 10V of Trizol and frozen at -80C.

value: L3 larvae


title: BIRD1_But2


title: CB5362 L3 Rep2 RNAseq


title: TY2205 L3 Rep2 RNAseq


title: TY2205 L3 Rep1 RNAseq

value: mRNA was purified from .5-10 ug of total RNA using Sera-Mag Oligo(dT) beads (ThermoScientific). cDNA preparation was done in the presence of dUTP to prepare stranded RNA-seq libraries as in PMID:19620215


title: CB5362 L3 Rep3 RNAseq


title: Blinc1-KO Rep2

city: Aurora

first: Lori

last: Sussel

state: CO

address: 1775 Aurora Ct.

institute: University of Colorado

data_processing:
RTA (Illumina) was used for base calling
CASAVA (version 1.8.2) was used for converting BCL to fastq format, coupled with adaptor trimming
Reads were mapped  to a reference genome (Mouse: UCSC/mm9) using Tophat (Trapnell C et al 2009 Bioinformatics) (version 2.0.4) with 4 mismatches (--read-mismatches = 4) and 10 maximum multiple hits (--max-multihits = 10).
Relative abundance (aka expression level) of genes and splice isoforms was determined using cufflinks (Trapnell C et al 2010 Nature Biotech) (version 2.0.2) with default settings.
Differentially expressed genes were calculated using Deseq (Anders S et al 2010 Genome Biology).
Genome_build: mm9
Supplementary_files_format_and_content:
csv-files includse FPKM values for all genes/isoforms in a sample
xls-file includes DeSeq analysis for all samples (Differentially expressed gene analysis)

extract_protocol:
Whole pancreas were dissected and total RNA was isolated using QIAGEN RNeasy Mini kits. Libraries were prepared from total RNA (RIN>8) with the TruSeq RNA prep kit (Illumina).
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain background

value: mixed; c57bl/6 and 129sv

value: e15.5 embryo

value: pancreas

tag: rnaseq id


title: Blinc1-Het Rep3


title: Blinc1-KO Rep1


title: Blinc1-Het Rep2


title: WT Rep3


title: WT Rep1


title: Blinc1-Het Rep1


title: RNA_14_Mouse_FoB_WT

city: Jerusalem

first: Howard

last: Cedar

address: Ein Kerem Campus

country: Israel

institute: The Hebrew University

data_processing:
RNA-seq 50 bp single-end reads were obtained from the Hiseq 2500 and analyzed by TopHat2.  Differential expression between male and female mice was analyzed by CuffDiff v2.1.1 (using default parameters, q-value<=0.01).
genome build: mm9
processed data files format and content: gene_exp.diff.gz (contains the expression data as processed by Cuffdiff)

source_name: Follicular B cells

growth_protocol: B cells in   different differentation stages were isolated from C57/bl wild type or inducible knockout for Tet2 and Tet3 under the MB1 promoter

extract_protocol:
RNA was isolated using miRNeasy (Qiagen).
TruSeq RNA Sample Preparation Kit v2 (Illumina) was used for Library preparation.

tag: gender

value: male

tag: age

value: 6 weeks

value: NORMAL


title: RNA_20_Mouse_FoB_TET3KO

value: female

value: 7 weeks


title: RNA_22_Mouse_FoB_TET2_3KO


title: RNA_19_Mouse_FoB_TET3KO


title: RNA_18_Mouse_FoB_TET2KO


title: RNA_23_Mouse_FoB_TET2_3KO


title: RNA_24_Mouse_FoB_TET2_3KO


title: RNA_17_Mouse_FoB_TET2KO


title: RNA_15_Mouse_FoB_WT


title: RNA_21_Mouse_FoB_TET3KO


title: CB5362 L3 Rep1 RNAseq


title: RNA_13_Mouse_FoB_WT

value: 8 weeks


title: RNA_3_Mouse_ProB_WT

source_name: Pro B cells


title: RNA_1_Mouse_ProB_WT


title: RRBS_22_Mouse_FoB_TET2_3KO

data_processing:
100 bp paired-end sequencing reads from RRBS were obtained using the Hiseq 2500.  Adapter trimming and quality filtering was performed with the trim_galore software (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)23using default parameters for RRBS analysis.  BSMAP24 was used for read alignment (using genome builds mm9 and hg19) and extraction of single base-resolution methylation levels.  100 bp tiles and DMRs were calculated with the MethylKit package25 using a minimum coverage of 10 per tile, methylation difference of 25%, and q value <= 0.01.  In order to increase sensitivity, DMRs were further filtered for multiple alignments in the genome using blat26 and excluding tiles that were mapped more than once.  The statistical significance of the DMR set was calculated by repeating the above steps on random permutations of male and female samples.  For each permutation the number of DMRs was calculated and a p-value was obtained by measuring the quantile of the original DMRs.  For tissues with at least 8 samples (Liver and Muscle) we included the condition that the difference between the second highest and second lowest values will not exceed 30%.
genome build: mm9
processed data files format and content: 100bpTiles_RRBS_Mouse.txt.gz (contains 100bp tiles methylation data of all the RRBS Mouse samples)

extract_protocol:
DNA was isolated from isolated B-cells and incubated in lysis buffer (25  mM Tris-HCl (pH  8), 2  mM EDTA, 0.2% SDS, 200  mM NaCl) supplemented with 300 μg/ml proteinase K (Roche) followed by phenol:chloroform extraction and ethanol precipitation.
RRBS libraries were prepared as described (Boyle, P. et al. 2012)


title: RRBS_23_Mouse_FoB_TET2_3KO


title: RNA_2_Mouse_ProB_WT


title: RNA_10_Mouse_ProB_TET2_3KO


title: RNA_16_Mouse_FoB_TET2KO


title: RRBS_16_Mouse_FoB_TET2KO


title: RRBS_19_Mouse_FoB_TET3KO


title: RRBS_15_Mouse_FoB_WT


title: RRBS_20_Mouse_FoB_TET3KO


title: WT Rep2


title: RRBS_21_Mouse_FoB_TET3KO


title: Blinc1-KO Rep3


title: RRBS_24_Mouse_FoB_TET2_3KO


title: RRBS_17_Mouse_FoB_TET2KO


title: RRBS_14_Mouse_FoB_WT


title: RRBS_18_Mouse_FoB_TET2KO


title: RRBS_8_Mouse_ProB_TET3KO


title: RRBS_13_Mouse_FoB_WT


title: RRBS_2_Mouse_ProB_WT


title: RRBS_12_Mouse_ProB_TET2_3KO


title: RRBS_11_Mouse_ProB_TET2_3KO


title: RRBS_5_Mouse_ProB_TET2KO


title: RRBS_6_Mouse_ProB_TET2KO


title: RRBS_1_Mouse_ProB_WT


title: RRBS_10_Mouse_ProB_TET2_3KO


title: RRBS_9_Mouse_ProB_TET3KO


title: RRBS_7_Mouse_ProB_TET3KO


title: RRBS_3_Mouse_ProB_WT


title: RRBS_4_Mouse_ProB_TET2KO


title: ST_RNA-seq_Rep5

city: Chicago

first: Yingming

last: Zhao

state: IL

address: 929 East 57th St. W410

department: The Ben May Department for Cancer Research

institute: University of Chicago

data_processing:
Basecalls are performed using CASAVA version 1.8.2
Reads are aligned using TopHat version 2.0.0 and bowtie2 version 2.0.0-beta6, with default parameters.
Mapped reads are summarized for each gene using HTSeq framework version 0.6.1 with command line "htseq-count --stranded=no".
Differentially exprssed genes are identified using edgeR version 3.6.4 with parameters FDR = 0.05 & log2 fold change >= 0.5, after retaining only the genes of which cpm > 1 in at least five samples.
Genome_build: mm10
Supplementary_files_format_and_content: HTSeq output is a tab-limited text file with two columns, gene name and number of reads mapping to each gene.

extract_protocol:
Total RNAs were prepared by following protocols from RNeasy Mini Kit (QIAGEN INC, Valencia, CA).
RNA-seq libraries were constructed according to manufacturer instructions using TruSeq Stranded Total Sample Preparation kit (Illumina).

treatment_protocol: 5 pairs of male C57BL/6 mice were either fed with standard chow diet, or fasted (with free access to water) for 48 hours (9:00 am to 9:00 am).

value: 4 months

tag: sample group

value: liver


title: ST_RNA-seq_Rep4

value: fasted (with free access to water) for 48 h


title: ST_RNA-seq_Rep3


title: AL_RNA-seq_Rep4

value: fed with standard chow diet for 48hrs


title: ST_RNA-seq_Rep1


title: Input

city: Sendai

first: Akira

last: Uruno

state: Miyagi

address: 2-1 Seiryo-cho, Aoba-ku

country: Japan

institute: Tohoku University Graduate School of Medicine

data_processing:
Illumina bcl2fastq v2.15.0 software used for basecalling.
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie2 software.
Peaks were called using MACS version 1.4
Genome_build: mm9
Supplementary_files_format_and_content: bed files were generated using MACS.

source_name: C2C12 myotubes_input

growth_protocol: C2C12 myoblasts  were cultured in Dulbecco's modified Eagle's media (DMEM) containing 25 mmol/L glucose, 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. When the C2C12 myoblasts reached confluence, the culture medium was replaced with differentiation medium (DMEM supplemented with 2% horse serum), and the cells were cultured for an additional 72 h to facilitate myotube differentiation.

extract_protocol:
The cells were fixed with 1% formaldehyde for 10 min at room temperature and subsequently quenched with 0.125-M glycine. ChIP was performed with anti-Nrf2 antibody (Cell Signaling Technology; D1Z9C).The antibody incubation were pereformed overnight at 4 °C.
The DNA libraries were prepared from 2 ng of ChIP and input samples quantified with Qubit Fluorometer (Life Technologies), using Mondrian SP+ and Ovation SP Ultralow DR Multiplex System (TaKaRa).braries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: After 3 days of differentiation, C2C12 myotubes were treated with 100 nmol/L CDDO-Im (oleanolic acid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole) for 3 hours

tag: cell type

value: myoblast cell line

value: wild type

tag: treated with

value: 100 nmol/L CDDO-Im for 3 h


title: ST_RNA-seq_Rep2


title: C2C12_ChIP_2

source_name: C2C12 myotubes_ChIP

tag: cell line

tag: chip antibody

value: anti-Nrf2 antibody

tag: chip antibody vendor

value: Cell Signaling Technology

tag: chip antibody cat.#


title: AL_RNA-seq_Rep2


title: AL_RNA-seq_Rep3


title: AL_RNA-seq_Rep1


title: AL_RNA-seq_Rep5


title: H3K4me3_ST_ChIP-seq

data_processing:
Basecalls are performed using CASAVA version 1.8.2
Reads are aligned using bowtie version 0.12.7 with parameters "-q --sam -a -v 2 -m 1 -p 20".
Reads are extended by 200bp to their 3' directions and the number of reads mapping to each of non-overlapping 200bp windows across a genome are summarized.
Peaks are called using the MOSAiCS algorithm with FDR = 1e-10.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-limited text files have three columns, where three columns are chromosome ID, genomic coordinate, and number of reads mapping to each window of size 200bp starting from the genomic coordinate. Peak calling results are provide as BED files, where the scores are average ChIP counts.

source_name: liver

extract_protocol:
ChIP DNAs were prepared following the protocols from ChIP-IT® High Sensitivity kit (Active Motif, Carlsbad, CA).
ChIP-seq libraries were prepared following the Ovation Ultralow DR Multiplex System protocols (NuGEN Technologies INC, San Carlos, CA), followed by a size selection (200-700bp) step by SPRIselect beads (Beckman Coulter, Brea, CA).


title: H3K9ac_ST_ChIP-seq


title: H4K8bhb_ST_ChIP-seq

value: H4K8bhb (PTM Biolabs)


title: H3K4bhb_ST_ChIP-seq

value: H3K4bhb (PTM Biolabs)


title: H3K9bhb_ST_ChIP-seq

value: male

value: H3K9bhb (PTM Biolabs)


title: Input_ST_ChIP-seq


title: mouse_pancreas_miRNAs

city: Nanjing

first: Zhen

last: Zhou

state: Jiangsu

address: 22th,Hankou Road

department: School of Lifesciences

institute: Nanjing University

data_processing:
Illumina Casava1.6 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then aligned to pre-miRNA sequence in miRBase_v16
Sequenced reads with no more than 2 mismatches and 2 shifts were identified as one miRNA and formed the final mouse 14 tissues' miRNA expression profiles.
Genome_build: miRBase_v16
Supplementary_files_format_and_content: tab-delimited text files with miRNA names and their counts of all 14 mouse tissues

extract_protocol:
14 mouse tissues were harvested from 5 C57Bl/6J mouse. Total RNA was harvested using Trizol reagent. Total RNAs from 10 mouse were pooled together to build RNA libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: pancreas


title: H3K9ac_AL_ChIP-seq


title: mouse_colon_miRNAs

value: colon


title: mouse_lung_miRNAs

value: lung


title: mouse_ovary_miRNAs

value: ovary


title: mouse_cerebellum_miRNAs

value: cerebellum


title: mouse_heart_miRNAs

value: heart


title: H3K9bhb_AL_ChIP-seq

value: 4 months

value: liver


title: mouse_spleen_miRNAs

value: spleen


title: C2C12_ChIP_1

value: myoblast cell line

tag: genotype/variation

value: wild type


title: H3K4bhb_AL_ChIP-seq

tag: strain

value: fed with standard chow diet for 48hrs


title: mouse_uterus_miRNAs

value: uterus


title: H4K8bhb_AL_ChIP-seq

value: H4K8bhb (PTM Biolabs)


title: mouse_small_intestines_miRNAs


title: mouse_brain_miRNAs

value: brain


title: mouse_liver_miRNAs


title: Hep4_Rif

city: Indianapolis

first: Hai

last: Lin

address: 410 W 10th St

institute: IUPUI

data_processing:
Sequence reads were aligned with BFAST (v.0.6.4e) towards reference genome and junction library. Details refers to Breese and Liu, Bioinformatics, 2013, PMID: 23314324
Aligned reads were fitered with NGSUtils (Breese and Liu, Bioinformatics 2013), only unique mapped reads with no more than 2 mismatchs were kept.
Read counts and RPKM was calculated with NGSUtils based on RefSeq annotation.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include raw counts and RPKM values

source_name: primary hepatocyte_rifampin

growth_protocol: Human hepatocytes from seven different subjects were obtained from CellzDirect (Durham, NC). The primary human hepatocytes were grown on collagen coated , cultured in Williams' E medium without phenol red containing Primary Hepatocyte Maintenance Supplements (Life Technologies Corporation, Carlsbad, CA). Each culture from a different individual subject was treated as a biologic replicate (n = 7).

extract_protocol:
Trizol per manufacturer's (Invitrogen) protocol
RNA was fragmented with RNAaseIII, cDNA libraries constructed with SOLiD adaptors by reverse transcription

treatment_protocol: In vitro studies were performed within 72 to 120 hours from the time of hepatocyte isolation. The primary human hepatocytes were grown on collagen coated plates and treated with rifampin (10 µM) or vehicle (methanol, 0.01%) for 24 hours.

tag: treatment

value: rifampin

value: primary hepatocyte cells


title: mouse_kidney_miRNAs

value: kidney


title: Hep4_Ctrl

source_name: primary hepatocyte_vehicle

value: vehicle


title: mouse_oesophagus_miRNAs

value: oesophagus


title: CYC2_3.5 h_RNA-seq

city: Hefei

first: Xiaoyuan

last: Song

state: Anhui

address: Huangshan Road 443

department: School of Life Sciences

institute: University of Science and Technology of China

description: processed data file: CYC2vsWT Conj-3.5hr RNA-Seq.xlsx

data_processing:
Raw sequencing reads were trimmed by removing illumina adapter sequences and low quality bases
The STAR program with default parameters was used to map the sequenced reads to the reference genome and to find those that spanned exon-exon junctions
Transcripts were assembled using Cufflinks and gene expression were based on fragments per kilobase of exon model per million mapped reads (FPKM) value.
FPKM of different samples were compare by using Cuffdiff
Genome_build: Tetrahymena thermophila 2014 assembly genome from The Tetrahymena Genome Database (TGD; www.ciliate.org)
Supplementary_files_format_and_content: Cuffdiff output of WT and CYC2 KO at four stages (containing 'Gene Model Identifier' such as TTHERM_000138239 retrievable at TGD)

organism: Tetrahymena thermophila

source_name: CYC2 knockout T. Thermophila cells

growth_protocol: Tetrahymena thermophila strains were routinely cultured in super proteose peptone (1×SPP) medium (1% proteose peptone, 0.2% glucose, 0.1% yeast extract, and 0.003% EDTA ferric sodium salt) at 30°C with vigorous shaking (rotation speed usually at 180-220rpm), unless otherwise specifically requested.

extract_protocol:
Cells for each of the samples with the amount of approximately 1×107 (cells) for each samples were washed once with 1×PBS (centrifuge at 450×g for 2min) and spun down to pellet followed by immediate freezing in liquid nitrogen.The total RNA of the freezed cell samples were then extracted by Trizol lysis followed by chloroform and isopropanol treatment.
After extract total RNA and treated with DNase I, Oligo(dT) are used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA are fragmented. Then  cDNA  is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are selected for the  PCR  amplification. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time  PCR  System are used in quantification and qualification of the sample library. Then the library is sequenced using Illumina HiSeq 4000 .

treatment_protocol: For starvation, log phase cells were washed and resuspended in 10 mM Tris (pH 7.4) at 30°C without shaking for 24 hr unless specified otherwise.

value: cyc2p knock out

tag: time point

value: 3.5hrs after conjugation initiation


title: mouse_stomach_miRNAs

tag: tissue

value: stomach


title: H3K4me3_AL_ChIP-seq

value: 4 months

value: male


title: CYC2_3 h_RNA-seq

description: processed data file: CYC2vsWT Conj-3hr RNA-Seq.xlsx

value: 3hrs after conjugation initiation


title: Hep1_Rif


title: CYC2_2.5 h_RNA-seq

description: processed data file: CYC2vsWT Conj-2.5hr RNA-Seq.xlsx

value: 2.5hrs after conjugation initiation


title: WT_3.5 h_RNA-seq

source_name: wild type T. Thermophila cells

value: wild type


title: Hep1_Ctrl


title: 5Aug10_Ctrl


title: WT_3 h_RNA-seq


title: 4Jul10_Rif


title: WT_2.5 h_RNA-seq


title: 5Aug10_Rif


title: Input_AL_ChIP-seq

value: fed with standard chow diet for 48hrs

value: liver


title: WT_2 h_RNA-seq

description: processed data file: CYC2vsWT Conj-2hr RNA-Seq.xlsx

value: 2hrs after conjugation initiation


title: 4Jul10_Ctrl


title: shUSP51_H2Bub_2

city: Smithville

first: Sharon

middle: Roth

last: Dent

state: Texas

address: 1808 Park Road 1C

institute: Univ Texas M.D. Anderson Cancer Center

data_processing:
Basecalling was done by Illumina analysis pipeline CASAVA (version 1.8.2).
Sequenced DNA reads were mapped to human genome hg19 using bowtie (version 1.1.2) with at most 2 mismatches allowed and only the reads that were mapped to unique position were retained. To avoid PCR bias, for multiple reads that were mapped to the same genomic position, only one copy was retained for further analysis.
Each read was extended by 150 bp (expected fragment size) from its 5’ end with respect to the strand orientation and the number of reads overlapping the gene body of each known transcript from RefSeq database (downloaded from UCSC Genome Browser on March 6, 2013) was counted. For the transcripts that share the same transcription start site and end site, only one representative transcript was used. Transcripts with less than 10 fragments in all the samples were removed.
The differential H2Bub signal enrichment between conditions was statistically assessed by R/Bioconductor package edgeR (version 3.6.2). The total number of tags in intragenic regions for each sample was passed to edgeR as the original library size. Genes that have transcripts with FDR (false discovery rate) ≤ 0.05 were called significant.
Genome_build: hg19
Supplementary_files_format_and_content: The processed files record the differential H2Bub signal result by edgeR on all the genes in ATXN7L3/USP22/USP27x/USP51 depleted samples comparing to control. The columns are: gene_symbol(gene symbol); sig1(normalized signal of control sample); sig2(normalized signal of ATXN7L3/USP22/USP27x/USP51 depleted sample); log2ratio(log2 ratio of H2Bub signal to control); pvalue(p-value); FDR(false discovery rate);

growth_protocol: Cells were were maintained at 37C and 5%CO2 in HyClone DMEM-H medium, supplemented with 10% FBS and 1% Pen-Strep.

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Antibody-histone-DNA complexes were harvested using protein G magnetic dynabeads (ThermoFisher Scientific, Ref-10004D, Lot-163538810)
CHIP libraries were prepared using a modified Kapa LTP Library Preparation kit (KAPA Biosystems, Wilmington, MA) protocol for Illumina Platforms. 10ng of sheared DNA was used to repair the ends of the damaged fragments using a proprietary master mix. The resulted blunted fragments had to be 3’ A-Tailing using a proprietary mixture of enzymes to be able to ligate to the specific NexTflex adaptors from Bioo Scientific (Bioo Scientific, Austin, TX). For each of the steps (End Repair, 3’A Tailing, and Adaptor Ligation was followed by column clean up (Qiagen, Valencia, CA). After adapter ligation, DNA enrichment was performed using Kapa HiFi Hot Start Ready mix PCR, and a cocktail of primer mix. (1 cycle at 98°C for 45 seconds; 4 cycles of 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; 1 cycle at 72°C for 1 minute), and purified with AmpureXP beads (Beckman Coulter, Indianapolis. IN).The final library validation was checked using the 2200 TapeStation from Agilent Technologies (Agilent, Santa Clara, CA). Libraries were quantified using a Kapa Library Quantification Kit (KAPA Biosystems) and loaded in a flow cell for cluster generation using the Illumina cBOT (Illumina, San Diego, CA) at final concentration of 10pM

treatment_protocol: Cells were transduced with lentiviral vectors, expressing the indicated shRNA and stable clones were selected in medium containing puromycin (2.5ug/ml). After the selection, cells were maintained in medium without puromycin.

value: Cultured MCF7 cells (ATCC HTB-22)

value: luminal epithelial

value: depleted for USP51

value: Anti-Ubiquityl-Histone H2B Antibody, clone 56 (Millipore, catalog# 05-1312, lot# 2654212)


title: 16Jun10_Rif

value: rifampin

value: primary hepatocyte cells


title: 16Jun10_Ctrl

value: vehicle


title: shUSP51_H2Bub_1


title: shUSP27x_H2Bub_1

value: depleted for USP27x


title: shUSP22_H2Bub_1

value: depleted for USP22


title: shATXN7L3_H2Bub

value: depleted for ATXN7L3


title: 30Jul10_Ctrl


title: shUSP27x_H2Bub_2


title: Hep2_Ctrl


title: pLKO_H2Bub_2

value: control (expressing pLKO)


title: pLKO_H2Bub_1


title: shUSP22_H2Bub_2


title: CYC2_2 h_RNA-seq

value: cyc2p knock out


title: pGIPZ_H2Bub

value: control (expressing pGIPZ)


title: shUSP51_2

data_processing:
Basecalling was done by Illumina analysis pipeline CASAVA (version 1.8.2).
The reads were mapped to human genome (hg19) by TopHat (version 2.0.7)
The number of fragments in each known gene from RefSeq database (downloaded from UCSC Genome Browser on March 6, 2013) was enumerated using htseq-count from HTSeq package (version 0.5.3p9)
Genome_build: hg19
Supplementary_files_format_and_content: Number of fragments in each gene

extract_protocol:
Total RNA from sub confluent (~75%) MCF7 cells expressing non-targeting shRNA or shRNA targeting either USP22, USP27x or USP51 was isolated using an RNeasy kit (Qiagen cat# 74134). The purified RNA was then treated with TUBRO DNase. (Ambion cat. # A1907) and used for library preparation.
RNA-Seq sequencing libraries were made using TruSeq RNA library preparation kit (Illumina)


title: shUSP22_1


title: shUSP27x_2


title: pLKO_1

source_name: pLKO


title: pGIPZ_1

source_name: pGIPZ


title: PHTd_3_18

city: Columbia

first: Toshihiko

last: Ezashi

state: MO

address: 240a CS Bond Life Sciences Center

department: Animal Sciences

institute: University of Missouri-Columbia

description: differentiated primary trophoblast

data_processing:
The libraries were sequenced as single-end, 50 base read length on an Illumina HiSeq 2500 (Illumina Inc.) with an average read count of 21-28 million reads per sample.
Initial base calling and quality filtering of the mRNA-Seq reads generated with the Illumina analysis pipeline (fastQ format) was performed using the FastxToolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and adapters trimmed using CutAdapt (https://code.google.com/p/cutadapt/).
Reads that mapped to the human mitochondrial genome or to Phix were removed using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml).
Reads were mapped (tophat v2.0.13; http://ccb.jhu.edu/software/tophat/index.shtml) to the reference genome (from Ensembl, v78; http://www.ensembl.org/index.html) Homo_sapiens.GRCh38.dna.primary_assembly.fa using Homo_sapiens.GRCh38.78.gtf as annotation.
Tests for differential expression were made using cuffdiff v2.2.1.
Genome_build: GRCh38
Supplementary_files_format_and_content: Cuffdiff output

source_name: term placenta

growth_protocol: Primary trophoblasts were isolated from third trimester placentas after Trypsin/Dispase/DNaseI digestion followed by purification on a Percoll gradient. Primary trophoblasts were cultured for 9h (undifferentiated) to 48h (differentiated)  in DMEM supplemented with 10% FBS (Fetal Bovine Serum), 20 mM HEPES pH 7.4, and a mixture of penicilin-streptomycin-fungizone. Cells were maintained at 37ºC/5% CO2.

extract_protocol:
Total RNA were prepared using miRNeasy Mini Kit following the manufacturer's instructions
Libraries were constructed following the Illumina’s protocol with reagents supplied in Illumina’s TruSeq mRNA stranded sample preparation kit.

value: placenta

value: male

tag: time in vitro culture

tag: date of tissue preparation


title: pGIPZ_2


title: PHTd_3_16

description: differentiated primary trophoblast


title: PHTu_3_14

description: undifferentiated primary trophoblast

value: 9 h


title: shUSP51_1


title: iTreg_CPI703_Input

city: Cambridge

first: Barbara

middle: M

last: Bryant

state: MA

address: 215 First Street

institute: Constellation Pharmaceuticals

data_processing:
50-nucleotide sequence reads were aligned to the hg19 genome using the BWA algorithm with default settings. Only reads that passed Illumina’s purity filter, aligned with no more than 2 mismatches and mapped uniquely to the genome, were used in subsequent analyses.
The aligned read files were sorted and duplicates removed using sort and rmdup functions from samtools version 0.1.18 (Li et al., 2009).
WIG files were generated using IGVTools (version 2.2.2) count function with a window size of 25 nucleotides, an extension of 100 beyond the 50-nucleotide read length, and genome hg19 (Robinson et al., 2011; Thorvaldsdottir et al., 2013). The WIG files were scaled assuming an effective genome length of 2.79B base pairs, so that the mean signal would be 1.0.
Genome_build: hg19
Supplementary_files_format_and_content: WIG files and corresponding TDF files suitable for visualization using the Integrative Genomics Viewer (IGV) available from the Broad Institute.

source_name: Induced regulatory T cells

growth_protocol: Leukopak samples were procured from the Biological Specialty Corporation (Colmar, PA) and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (GE Biosciences) density gradient centrifugation. Naive CD4+ CD45RA+ T cells were isolated from PBMCs to a purity > 98% using Miltenyi naive human T cell isolation kits (130-094-131, Miltenyi Biotech). Isolated cells were cultured at 10^6 cells/mL under Treg-polarizing conditions, using Human T-Activator CD3/CD28 Dynabeads® (11132D, Invitrogen), human TGFb at 10 ng/mL and human IL-2 at 10 U/mL (100-B and 202-IL, respectively; R&D Biosystems).

extract_protocol:
For ChIP-seq, cells were pelleted, fixed in 1% formaldehyde for 10 mins, lysed and sonicated. Chromatin samples were precleared with Protein A Dynabeads (Life Technologies), and incubated overnight at 40C with anti-H3K18 Ac (9675, Cell Signaling), anti-H3K27 Ac (ab4729, Abcam), and anti-H3K4 Me3 (Abcam ab8580). Chromatin-antibody complexes were precipitated using Protein A Dynabeads followed by washes in RIPA buffer and Tris/ EDTA. Samples were digested with RNAase A, treated with Proteinase K and 10% SDS, followed by cross-link reversal at 650C. DNA was purified using MinElute PCR purification kits (Qiagen).
DNA libraries for ChIP-seq were prepared using Ovation Ultralow DR Multiplex System kits (0330-32, NuGEN) followed by Illumina sequencing at the MIT BioMicro Center.

treatment_protocol: Naïve human CD4+ T cells were treated with 4 mM CPI703 or DMSO under Treg polarizing conditions (described above) for 4 days.

value: Treg cell


title: iTreg_DMSO_H3K27ac

value: DMSO

value: anti-H3K27ac (Abcam, catalog# ab4729, lot# GR132150-1)


title: iTreg_CPI703_H3K4me3

value: anti-H3K4me3 (EMD Millipore, catalog# 07-030, lot# 2477948)


title: PHTu_2_9

description: undifferentiated primary trophoblast


title: PHTd_2_10

description: differentiated primary trophoblast


title: iTreg_DMSO_Input


title: iTreg_DMSO_H3K4me3


title: shUSP22_2

value: Cultured MCF7 cells (ATCC HTB-22)

value: luminal epithelial


title: 48h infection

city: Hangzhou

first: Chu

last: ZhenJian

state: Zhejiang

address: Yuhangtang Road 388

department: College of Life Science

institute: Zhejiang University

data_processing:
Illumina HiSeqTM 2000 software used for basecalling.
Through quality test we remove low quality sequences, tag with N ratio>5%, empty reads and adaptor sequences then we got clean reads. Map the clean reads to the genome database of P. xylostella and B. bassiana by SOAPaligner/SOAP2
The data was normalized by reads per kilobase transcriptome per million mapped reads (RPKM)
genome build: Plutella xylostella: DBM_FJ_V1.1 and Beauveria bassiana: ASM28067v1.
We generating the RPKM data by RPKM = 10^6C divided by NL/10^3
C means the number of reads perfect match the gene
N means the total of reads
L means the base number of the gene
Supplementary_files_format_and_content: Table S1-S6 List of detected P. xylostella genes .xlsx
Supplementary_files_format_and_content: Table S10-S14. List of DEGs of P. xylostella genes.xlsx
Supplementary_files_format_and_content: Table S15-S16 List of different expressed genes of B. bassiana genes.xlsx
Supplementary_files_format_and_content: Table S7-S9 List of dected B.bassiana genes.xlsx

organism: Plutella xylostella

source_name: Infected larvae

growth_protocol: P. xylostella culture was maintained on cabbage plants in climate chambers at 25 ± 1 °C, 14 h of light and 10 h of darkness and 75 ± 10% relative humidity; The fungal strain B. bassiana ARSEF2860 from the RW Holley Center for Agriculture and Health (Ithaca, NY, USA) was preserved at 4°C on slants of Sabouraud dextrose agar (Qiu et al., 2014). Conidia harvested from cultures grown for 7 days at 25°C and 90% relative humidity were suspended in 0.02% Tween 80 (109 conidia/ml)

extract_protocol:
Total RNA was extracted from the P. xylostella using the RNAiso-Plus Reagent (TaKaRa). The cDNA libraries were prepared according to the manufacturer’s instructions (Illumina, San Diego, CA, USA)
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Third instar P. xylostella larvae were collected into Petri dishes as described above. At first, the sample of 48 hptI and its control were sprayed with 1 ml conidial suspension and 0.02% Tween 80, respectively. After 12 hours, the sample of 36 hptI and its control were sprayed with 1 ml conidial suspension and 0.02% Tween 80. In the same way the sample of 24 hptI and its control was handled. After 48 h of the first spray, approximately 200 living P. xylostella larvae were collected from each of the 6 treatments, respectively. All P. xylostella larvae were collected at the same time can eliminate the effect of host development on gene expression

tag: agent

value: Beauveria bassiana


title: PHTu_2_8

description: undifferentiated primary trophoblast

tag: gender


title: PHTd_3_17

description: differentiated primary trophoblast


title: shUSP27x_1


title: iTreg_DMSO_H3K18ac

value: anti-H3K18ac (Cell Signaling, catalog# 9675, lot# 3)


title: PHTu_2_7

description: undifferentiated primary trophoblast


title: pLKO_2


title: 36h infection


title: Treg_DMSO (SL95740)

description:
SL95740
3085-CN-0022
DMSO Treg

data_processing:
Sequencing was performed on an Illumina HiSeq 2000.
Tophat 1.4.1 was used to perform sequence alignment, with UCSC human (hg19) as the reference genome.
The Normalized counts of sequence reads (FPKM) mapped to annotated UCSC genes were determined using Cufflinks software.
The data was filtered to keep genes with at least one sample above a 50-read threshold. Samples with fewer than 10 reads FPKM are replaced with the average across samples of 10 reads FPKM, to generate the final gene counts for each sample and gene.
Data processing was performed by Ocean Ridge Biosystems.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files with sample annotations, Ensembl gene annotations and log2 FPKM expression values for each library / gene.

extract_protocol: RNA was isolated using Qiagen RNeasy Plus mini kits and sequenced at Ocean Ridge Biosciences.


title: iTreg_CPI703_H3K18ac


title: PHTu_3_15

description: undifferentiated primary trophoblast


title: 24h infection


title: PHTd_1_5

description: differentiated primary trophoblast

value: female


title: Treg_CPI703_4uM (SL95739)

description:
SL95739
3085-CN-0021
CPI-703 4uM Treg

tag: concentration


title: 48h control

data_processing:
Illumina HiSeqTM 2000 software used for basecalling.
Through quality test we remove low quality sequences, tag with N ratio>5%, empty reads and adaptor sequences then we got clean reads. Map the clean reads to the genome database of P. xylostella and B. bassiana by SOAPaligner/SOAP2
The data was normalized by reads per kilobase transcriptome per million mapped reads (RPKM)
genome build: Plutella xylostella: DBM_FJ_V1.1 and Beauveria bassiana: ASM28067v1.
Supplementary_files_format_and_content: Table S1-S6 List of detected P. xylostella genes .xlsx
Supplementary_files_format_and_content: Table S10-S14. List of DEGs of P. xylostella genes.xlsx
Supplementary_files_format_and_content: Table S15-S16 List of different expressed genes of B. bassiana genes.xlsx
Supplementary_files_format_and_content: Table S7-S9 List of dected B.bassiana genes.xlsx

source_name: Uninfected larvae


title: PHTd_2_12

description: differentiated primary trophoblast


title: PHTd_2_11

description: differentiated primary trophoblast


title: CD4_MRE

city: SAINT LOUIS

first: Daofeng

last: Li

address: 4515 McKinley Avenue

department: Genetics

institute: Washington University in St. Louis

data_processing:
Both MeDIP-seq and MRE-seq reads were aligned to human genome (hg19) with BWA
For MeDIP-seq, redundant reads were discarded
For MRE-seq, only reads with ends mapped to MRE restriction sites were kept
Alignment data were then processed by methylCRF to generate DNA methylation calls for each library
All methylCRF calls were combined into a table with rows representing CpGs and columsn representing DNA methylation level of a sample
Genome_build: hg19
Supplementary_files_format_and_content: text file containting DNA methylation methylCRF predictions

extract_protocol:
For MRE: 5 μg genomic DNA was isolated using Proteinase K. Parallel restriction enzyme digests were performed (HpaII, HinP1l,AciI, HpyCH4IV, New England Biolabs; Bsh1236I, Thermo Scientific), each with 1μg of DNA. Five units of enzyme per microgram DNA were added and incubated at 37 °C for a total of 6 hours. Digested DNA was precipitated with sodium acetate and ethanol, and 500 ng of each digest were combined into one tube. Combined DNA was size-selected by electrophoresis on a 1% agarose gel. A 100–300 bp gel slice was excised using a sterile scalpel and gel-purified using Qiagen QIAquick columns, eluting in 30 µl of Qiagen EB buffer.
Library construction was performed using the Illumina Genomic DNA Sample Kit (Illumina) with single-end adapters, following the manufacturer’s instructions with the following changes. For the end repair reaction, T4 DNA polymerase and T4 polynucleotide kinase were excluded and the Klenow DNA polymerase was diluted 1:5 in water and 1 µl used per reaction. For single-end oligo adaptor ligation, adapters were diluted 1:10 in water and 1 μl used per reaction. Fifteen cycles of PCR were performed using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (220-520 bp) was performed by electrophoresis in DNA was eluted in 36 μl EB buffer using Qiagen QIAquick columns. After cleanup with Qiagen MinElute columns, each library was examined by spectrophotometry (Nanodrop, Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent).2% agarose.

value: Blood

tag: sample type

tag: assay

value: MRE


title: PHTd_1_4

description: differentiated primary trophoblast


title: 36h control


title: 24h control


title: WholeBlood_MRE

value: whole blood


title: PHTu_1_3

description: undifferentiated primary trophoblast


title: SNcDKO ESC, replicate 3 [RNA-seq]

city: Yokohama

first: Takaho

middle: A.

last: Endo

state: Kanagawa

address: 1-7-22 Suehiro, Tsurumi

department: IMS

institute: RIKEN

data_processing:
Basecalls performed using Illumina offline basecaller (OLB) version 1.9.4
Sequences were aligned on mouse genome (mm9) using tophat2 (version 2.1.0) with option "--no-coverage search".
Gene expression was estimated using cufflinks (version 2.1.0) with default options.
Genome_build: mm9
Supplementary_files_format_and_content: plain text containig FPKM (fragments per kilobase exon per million reads) values.

source_name: SNcDKO ESC

growth_protocol: Standard condition for ES cells.

extract_protocol:
Genomic DNA was isolated using QIAGEN All-prep kit
Libraries were prepared according to NEB's instructions accompanying the NEBNext® ChIP-seq Library Prep Reagent set for Illumina.  Adapter ligated DNA was PCR amplified with Illumina primers for 15 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Hi-Seq 1000 following the manufacturer's protocols.

tag: strain background

value: Setdb1 and Np95 double (flox/flox) conditional alleles; CreERT2 transgene

value: With 4-OHT (day 6)


title: CD4_MeDIP

extract_protocol:
For MeDIP-seq, 5 μg genomic DNA was isolated using Proteinase K. DNA was then sonicated to ~100–500 bp with a Bioruptor sonicator (Diagenode). Sonicated DNA was end-repaired, A-tailed, and ligated to single-end or paired-end adapters following the standard Illumina protocol. After agarose size-selection to remove unligated adapters, 2–5 μg of adaptor-ligated DNA was used for each immunoprecipitation using a mouse monoclonal anti-methylcytidine antibody (1 mg ml−1, Eurogentec. Catalog No. BI-MECY-0100). For this, DNA was heat-denatured at 95 °C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μl primary antibody per microgram of DNA overnight at 4 °C with rocking agitation in 500 μl immunoprecipitation buffer (10 mM sodium phosphate buffer, pH 7.0, 140 mM NaCl, 0.05% Triton X-100). To recover the immunoabsorbed DNA fragments, 4 μl of rabbit anti-mouse IgG secondary antibody (2.5 mg ml−1, Jackson Immunoresearch. Catalog No. 315-005-008) and 100 μl Protein A/G beads (Pierce Biotechnology. Catalog No. PI20421) were added and incubated for an additional 2 h at 4 °C with agitation. After immunoprecipitation a total of six immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg ml−1proteinase K for 2 h at 55 °C and then allowed to cool to room temperature. MeDIP DNA were purified using Qiagen MinElute columns and eluted in 16 μl EB (Qiagen).
Thirteen cycles of PCR were performed on 5 μl of the immunoprecipitated DNA using the single-end or paired-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (220-520 bp) was performed by electrophoresis in DNA was eluted in 36 μl EB buffer using Qiagen QIAquick columns. After cleanup with Qiagen MinElute columns, each library was examined by spectrophotometry (Nanodrop, Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent).2% agarose.

value: MeDIP


title: PHTu_1_2

description: undifferentiated primary trophoblast

value: placenta


title: PHTu_1_1

description: undifferentiated primary trophoblast

value: 9 h


title: SNcDKO ESC, replicate 1 [RNA-seq]


title: ESCu

description: processed data files: ESCd_lt40_over_ESCu.cuffdiff.xlsx, ESCd_40-70_over_ESCu.cuffdiff.xlsx, ESCd_gt70_over_ESCu.cuffdiff.xlsx

data_processing:
The libraries were sequenced as single-end, 50 base read length on an Illumina HiSeq 2500 (Illumina Inc.) with an average read count of 21-28 million reads per sample.
Initial base calling and quality filtering of the mRNA-Seq reads generated with the Illumina analysis pipeline (fastQ format) was performed using the FastxToolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and adapters trimmed using CutAdapt (https://code.google.com/p/cutadapt/).
Reads that mapped to the human mitochondrial genome or to Phix were removed using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml).
Reads were mapped (tophat v2.0.13; http://ccb.jhu.edu/software/tophat/index.shtml) to the reference genome (from Ensembl, v78; http://www.ensembl.org/index.html) Homo_sapiens.GRCh38.dna.primary_assembly.fa using Homo_sapiens.GRCh38.78.gtf as annotation.
Tests for differential expression were made using cuffdiff v2.2.1.

source_name: H1 hESC of 8 days culture with MEF-CM+FGF2 cultured in parallel with above BAP treated cells

extract_protocol:
RNAs were extracted with Trizol (Thermo) and 1-bromo-3-chloro-propane (Sigma).
Libraries were constructed following the Illumina’s protocol with reagents supplied in Illumina’s TruSeq mRNA stranded sample preparation kit.

tag: source cell line

value: H1 (NIH code WA01)

tag: cell type

value: ESC derived from human male blastocysts (H1, NIH code WA01)

tag: treated with

value: untreated (MEF-CM+FGF2 culture)

tag: age

value: 8-day culture


title: SNcDKO ESC, replicate 2 [RNA-seq]

tag: genotype/variation


title: Tonsil_2_MRE

value: Tonsil


title: ESCd_lt40

description: processed data files: ESCd_lt40_over_ESCu.cuffdiff.xlsx, ESCd_40-70_over_ESCd_lt40.cuffdiff.xlsx, ESCd_gt70_over_ESCd_lt40.cuffdiff.xlsx

source_name: Trophoblasts of less than 40 μm fraction generated with BAP treatment of H1 hESC for day 8

value: BAP-treatment

tag: cell fraction

value: Trophoblasts of less than 40 μm


title: SN wt ESC, replicate 3 [RNA-seq]

source_name: SN wt ESC

value: Without 4-OHT (day 0)


title: Tonsil_2_MeDIP


title: Tonsil_1_MRE


title: PHTd_1_6

description: differentiated primary trophoblast

value: female


title: ESCd_40-70

description: processed data files: ESCd_40-70_over_ESCu.cuffdiff.xlsx, ESCd_40-70_over_ESCd_lt40.cuffdiff.xlsx, ESCd_gt70_over_ESCd_40-70.cuffdiff.xlsx

source_name: STB of its size between 40 and 70 μm collected at day 8 BAP treatment of H1 hESC

value: STB of its size between 40 and 70 μm


title: Granulocyte_6_MRE

value: Blood

value: Granulocyte

value: MRE


title: SDcDKO ESC, replicate 2 [RNA-seq]

source_name: SDcDKO ESC

value: Setdb1 and Dnmt1 double (flox/flox) conditional alleles; CreERT2 transgene

value: With 4-OHT (day 6)


title: SN wt ESC, replicate 2 [RNA-seq]

value: Setdb1 and Np95 double (flox/flox) conditional alleles; CreERT2 transgene


title: Tonsil_1_MeDIP

value: MeDIP


title: ESCd_gt70

description: processed data files: ESCd_gt70_over_ESCu.cuffdiff.xlsx, ESCd_gt70_over_ESCd_lt40.cuffdiff.xlsx, ESCd_gt70_over_ESCd_40-70.cuffdiff.xlsx

source_name: STB of larger 70 μm collected at day 8 BAP treatment of H1 hESC

value: H1 (NIH code WA01)

value: STB of larger 70 μm


title: Np95cKO ESC, replicate 2 [RNA-seq]

source_name: Np95cKO ESC

value: Np95 (flox/flox) conditional alleles; CreERT2 transgene


title: Granulocyte_4_MeDIP


title: Granulocyte_5_MeDIP


title: Granulocyte_4_MRE


title: SDcDKO ESC, replicate 1 [RNA-seq]


title: Granulocyte_6_MeDIP


title: SD wt ESC, replicate 2 [RNA-seq]

source_name: SD wt ESC


title: WholeBlood_MeDIP

value: whole blood


title: Granulocyte_5_MRE


title: Np95cKO ESC, replicate 1 [RNA-seq]


title: Np95 wt ESC, replicate 2 [RNA-seq]

source_name: Np95 wt ESC


title: SN wt ESC, replicate 1 [RNA-seq]


title: Granulocyte_2_MRE


title: Granulocyte_3_MRE


title: Granulocyte_1_MRE


title: SD wt ESC, replicate 1 [RNA-seq]


title: Granulocyte_2_MeDIP


title: Granulocyte_3_MeDIP


title: Granulocyte_1_MeDIP


title: Np95 wt ESC, replicate 1 [RNA-seq]


title: Dnmt1cKO ESC, replicate 3 [RNA-seq]

source_name: Dnmt1cKO ESC

value: Dnmt1 (flox/flox) conditional alleles; CreERT2 transgene


title: Dnmt1cKO ESC, replicate 4 [RNA-seq]


title: Dnmt1cKO ESC, replicate 2 [RNA-seq]


title: CD14_Monocyte_4_MRE

value: CD14 monocyte


title: Dnmt1 wt ESC, replicate 2 [RNA-seq]

source_name: Dnmt1 wt ESC


title: CD14_Monocyte_4_MeDIP


title: Dnmt1 wt ESC, replicate 1 [RNA-seq]


title: DNcDKO ESC, replicate 1 [RNA-seq]

source_name: DNcDKO ESC

value: Dnmt1 and Np95 double (flox/flox) conditional alleles; CreERT2 transgene


title: DNcdKO ESC, replicate 2 [RNA-seq]

source_name: DNcdKO ESC


title: CD14_Monocyte_3_MRE


title: DN+Myc-Np95 cDKO ESC [RNA-seq]

source_name: DN+Myc-Np95 cDKO ESC

value: Dnmt1 and Np95 double (flox/flox) conditional alleles; exogenous Myc-tagged Np95 expressed; CreERT2 transgene


title: DN+Myc-Np95 wt ESC [RNA-seq]

source_name: DN+Myc-Np95 wt ESC

value: Without 4-OHT (day 0)


title: CD14_Monocyte_1_MRE


title: CD14_Monocyte_2_MeDIP


title: CD14_Monocyte_2_MRE


title: CD14_Monocyte_1_MeDIP


title: DN wt ESC, replicate 2 [RNA-seq]

source_name: DN wt ESC


title: Dnmt1cKO ESC, replicate 1 [RNA-seq]


title: CD8_Memory_MRE

value: CD8 memory


title: GATA3 ChIP total thymocytes replicate 1

city: Ghent

first: Inge

last: Van de Walle

address: De Pintelaan 185, blokA 4th floor

country: Belgium

institute: UGent

data_processing:
Illumina Casava1.7 software used for basecalling.
reads were aligned with Bowtie
peaks were called using MACS2 using input as control
Genome_build: hg19
Supplementary_files_format_and_content: bed files with peak and summit location, xls file with MACS2 output

source_name: Total child thymus

growth_protocol: Not applicable: freshly isolated primary cells were used

extract_protocol:
DNA was purified by phenol/chloroform extraction.
ChIP DNA were purified and end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3â 'A' base for ligation of Illumina's adapters which have a single 3' overhanging 'T' base. After adapter ligation DNA was PCR amplified with Illumina primers for 25 cycles, and the product was selected for 200~300 bp (insert plus adaptor and PCR primer sequences) by gel electrophoresis and extraction (QIAex II, Qiagen). The purified DNA was subjected for a second round of PCR amplification for 10 cycles. Libraries were sequenced on the Genome Analyzer II following the manufacturer's protocols.

treatment_protocol: The ChIP-protocol was performed as described (Zhang et al. 2012). In brief, 50*106 total thymocytes were cross-linked with 1% formaldehyde at 37°C for 10 min and the cross-linking reaction was quenched with glycine (125 mM final concentration, Sigma-Aldrich, G-8790). Cells were lysed using SDS lysis buffer and chromatin was fragmented to 200-600 bp fragments by sonication. Chromatin immunoprecipitation was performed by overnight incubation with 2,5 μg of control or anti-GATA3 antibody (Santa-Cruz Biotechnology, sc-268) and bound DNA was isolated the next day using anti-mouse secondary antibody-coupled magnetic beads (Dynabeads) following 1 hour incubation. Enriched chromatin fragments were eluted from the beads, followed by reverse cross-linking by incubation at 65°C for at least 6 hours.

value: thymus


title: CD8_Memory_MeDIP

value: Blood


title: H3K27Ac_ChIPSeq

city: Perth

first: Zhaoyu

state: Western Australia

address: QE II, M block, 225C

department: School of Biomedical Sciences

institute: University of Western Australia

data_processing:
ChIP-Seq reads were aligned to human genome assembly hg19 using bowtie 2.1.0 with default parameters.
Peaks were called by using HOMER v4.2 with "-style histone" to its input and default parameters.
Genome_build: GRCh37 (hg19 )
Supplementary_files_format_and_content: Peak files (.bed) were generated using HOMER.

source_name: T-47D, H3K27Ac ChIP

growth_protocol: T-47D cancer cells were grown to 70-80% confluence in DMEM (Gibco) supplemented with 10% FBS (Hyclone) in 100 mm dishes.

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared according to ChIP-Seq library preparation kit (NEB) and sequenced with the Illumina HiSeq 2000 analyzer at Mayo Clinic.

tag: cell line

value: breast cancer cell line

tag: chip antibody

value: H3K27ac (Abcam ab4729)


title: H3K4me2_ChIPSeq

source_name: T-47D, H3K4me2 ChIP

value: H3K4me2 (Millipore 07-030)


title: Input DNA

source_name: T-47D, input


title: H3K27me3_ChIPSeq

source_name: T-47D, H3K27me3 ChIP

value: H3K27me3 (Cell Signaling 9733S)


title: DN wt ESC, replicate 1 [RNA-seq]

tag: time point


title: GATA3 ChIP total thymocytes replicate 2

tag: tissue


title: H3K9me3_ChIPSeq

source_name: T-47D, H3K9me3 ChIP

value: H3K9me3 (Abcam ab8898)


title: input sample total thymocytes


title: CD14_Monocyte_3_MeDIP

value: MeDIP


title: Thymus_14

first: Milan

last: Radovich

state: Indiana

address: 980 W. Walnut St, Room C312

institute: Indiana University School of Medicine

description:
M14
mir_RPKM.txt
gene_RPKM.txt

data_processing:
Basecalling performed using SOLiD system.  Output (.csfasta and .qual) files were converted to XSQ Files.
Reads were mapped to hg19 using LifeScope 2.5.1
RPKM values were generated using ParTek Genomics Suite
Genome_build: hg19
Supplementary_files_format_and_content: mir_RPKM.txt contains miRNA precursor RPKM values and gene_RPKM.txt contains RefSeq gene expression RPKM

source_name: Normal Thymus

extract_protocol:
Ambion Ribopure Extraction Kit
Total RNA-Seq Kit (Life Technologies)
RNA-Seq including miRNA precursors

tag: disease state

value: Normal

tag: who subtype


title: Thymus_13

description:
M13
mir_RPKM.txt
gene_RPKM.txt

source_name: Thymic neoplasm

value: Tumor


title: Brain_GerminalMatrix_MRE

value: Brain Germinal Matrix

value: MRE


title: Thymus_11

description:
M11
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_7

description:
M7
mir_RPKM.txt
gene_RPKM.txt

value: A


title: Thymus_8

description:
M8
mir_RPKM.txt
gene_RPKM.txt


title: H2AZ_ChIPSeq

source_name: T-47D, H2A.Z ChIP

value: H2A.Z (Abcam ab4174)


title: Thymus_6

description:
M6
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_5

description:
M5
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_10

description:
M10
mir_RPKM.txt
gene_RPKM.txt


title: H3K36me3_ChIPSeq

source_name: T-47D, H3K36me3 ChIP

value: H3K36me3 (Active Motif 61101)


title: Thymus_9

description:
M9
mir_RPKM.txt
gene_RPKM.txt

value: A/B


title: Thymus_16

description:
M16
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_4

description:
M4
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_2

description:
M2
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_1

description:
M1
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_3

description:
M3
mir_RPKM.txt
gene_RPKM.txt


title: Thymus_15

description:
M15
mir_RPKM.txt
gene_RPKM.txt


title: MM1S_DMSO_4

status: Public on Apr 05 2016

city: Oxford

first: Udo

last: Oppermann

state: Oxfordshire

address: Botnar Research Centre, Nuffield Orthopaedic Centre, Windmill Road

department: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences

country: United Kingdom

institute: University of Oxford

data_processing:
Libraries were sequenced on the Illumina NextSeq 500 and Basecalling and demultiplexing performed by their standard pipelines
Adaptors and low quality reads were trimmed using Trim_galore version 0.40
Aligned to GRCh37 using Bowtie v 1.1.2 options -m3 -y --best
Peaks called using macs2 v 2.1.0 and the --gapped option
Homer v4.8 used to create tag directories of all samples and produced bedgraph data normalised to 10^7 reads.
Genome_build: GRCh37
Supplementary_files_format_and_content: bedgraph.gz - coveraged data in bedgraph format noramlised to 10e7 reads.  Gapped Peaks file;  is in BED12+3 format which contains both the gapped region and narrow peaks. The 5th column is 10*-log10qvalue, to be more compatible to show grey levels on UCSC browser. Tht 7th is the start of the first narrow peak in the region, and the 8th column is the end. The 9th column should be RGB color key, however, we keep 0 here to use the default color, so change it if you want. The 10th column tells how many blocks including the starting 1bp and ending 1bp of gapped regions. The 11th column shows the length of each blocks, and 12th for the starts of each blocks. 13th: fold-change, 14th: -log10pvalue, 15th: -log10qvalue.

source_name: MM1S Multiple Myeloma cells_DMSO

growth_protocol: Cells were grown under standard conditions in RMPI with 10% FGS and 2mmol glutamine supplemented

extract_protocol:
Cells were fixed in 1% formaldehyde for 8 minutes at room temperature, sonicated on a Bioruptor Pico, incubated for an hour with pre-blocked protein A seppharose beads before being incubated with 1ug of antibody overnight at 4 degrees.  The lysate was allowed to incubate with protein A beads before being washed and subejcted to proteinase K digestion
Libraries were constructed using NEBnext Ultra DNA library prep kit according to manufactures instructions

treatment_protocol: Cells were treated with either KDM5-C70 50uM for 7 days or vehicle control

value: Multiple Myeloma cell line

value: DMSO (vehicle control)

tag: chip antibody vendor

value: Millpore

tag: chip antibody cat. #

tag: chip antibody lot #


title: MM1S_KDM5C70_1

source_name: MM1S Multiple Myeloma cells_KDM5C70


title: MM1S_DMSO_5


title: MM1S_KDM5C70_3


title: MM1S_KDM5C70_2


title: MM1S_DMSO_2


title: MM1S_DMSO_1


title: Thymus_12

description:
M12
mir_RPKM.txt
gene_RPKM.txt

value: C


title: LAN6-Input_control-XL-MNase_ChIP

city: New York

first: Emily

last: Bernstein

address: 1470 Madison Avenue, 6th floor Rm 302

department: Oncological Sciences

institute: Mount Sinai School of Medicine

data_processing:
Basecalls performed using Illumina's Real-Time Analysis (RTA) Software version 1.18.64
Sequence reads were aligned to the GRCh37 (hg19) assembly using Bowtie v1.0.0 with the following parameters: -M 10 -k 1 -n 2 -l 70 --best
Redundant reads were eliminated using the macs2 (v2.1.0) filterdup option with default parameters.
The optimal normalization factor between ChIP and Input samples was calculated with the R NCIS package v1.0.1 using a shift size of 75bp.
Peak calling was performed with macs2 callpeak using the following options: --ratio X --broad --bdg -q 0.0005 --broad-cutoff 0.0005, where X is the normalization factor estimated by NCIS
ChIP/Input fold enrichment pileups were created with the macs2 bdgcmp tool using the -m FE option and converted to bigWig files using the bedGraphToBigWig program (v4) from the UCSC binaries.
All peaks overlapping with the Encode blacklisted regions were eliminated.
Genome_build: GRCh37
Supplementary_files_format_and_content:
- ChIP/Input fold enrichment bigWig file obtained with macs2 bdgcmp, as described.
- Bed file with significant peaks from macs2

growth_protocol: LAN6 cells were growth in 15cm plates at maximum confluency of ~10x10^6 cells per plate in RPMI + 10%FBS + 1x Penicillin/Streptomycin (gibco). 20 million cells were used per ChIP

extract_protocol:
XL-MNase ChIP: Cross-linked MNase ChIP was performed using the Cell Signaling SimpleChIP Enzymatic Chromatin IP Kit with Magnetic beads (cat. #9003). The vendor’s instructions were followed with some changes. Briefly: 20 million LAN6 cells were cross-linked for 10 minutes at room temperature in complete media, 100mM NaCl, 1mM EDTA pH 8.0, 50mM HEPES pH 8.0, 1% formaldehyde. Glycine was added to a final concentration of 125mM to stop the cross-linked reaction. The cells were washed once with cold PBS. Nuclei were obtained lysing the cells with 4ml of buffer A + DTT + Protease Inhibitor Cocktail (PIC) for 10 minutes at 4°C. Nuclei were washed with 3 ml of ice-cold buffer B + DTT. Nuclei were resuspended in 1ml of ice-cold buffer B + DTT, and digested with 8,000 gel U of Micrococal Nuclease from NEB (cat. #M0247S) at 37°C for 20 minutes. The reaction was stopped adding EDTA for a final concentration of 50nM and incubating on ice for 5 minutes. The nuclei were pelleted, and resuspended in 1ml of ChIP buffer + PIC + PMSF and incubated on ice for 10 min. The nuclei were split in two tubes and lysed by light sonication in a Bioruptor Twin (4 cycles, 20 sec ON/OFF, high power). The chromatin was cleared by centrifugation and a sample taken and processed (see bellow) to measure the concentration of the chromatin. 40ug of chromatin were diluted to 100ng/ul with ChIP buffer. 1% of the diluted chromatin was took as Input and the rest incubated with specific antibodies overnight at 4°C. 40 ul of ChIP-grade Magna ChIP protein A/G magnetic beads from Millipore (cat. #16-663) were added and incubated for 3 hours at 4°C. The beads were washed 3 times with 1ml of ChIP buffer, 1 time with 1ml of ChIP buffer + 350 mM NaCl, 1 time with 1 ml of LiCl buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 1% sodium deoxycholate, 1% Igepal, 250mM LiCl, not included in the kit) and 1 time with 1ml of TE. The beads and the Input samples were incubated in 100ul of Elution buffer for 30 min in a thermomixer at 65°C, 1200 rpm. The eluted material was incubated for 1 hour at 37°C 1ul of RNAse A (10mg/ml), followed with a 3 hour incubation at 55°C with 3ul of Proteinase K (20mg/ml). The cross-linked was reversed incubating the eluted material for 4-6 hours at 65°C. The DNA was purified using the MinElute PCR purification kit from Qiagen following the kit’s instructions and eluted in 30ul of Qiagen’s elution buffer. The amount and the pattern of the obtained DNA was determined using an Agilent 2100 Bionanalyzer High Sensitivity Kit.
25ng of ChIPed or Input DNA were processed for library preparation from each sample. All enzymes used are from NEB unless stated otherwise. 1. DNA was End Repaired in 50ul reaction containing 40ul of DNA, 5ul T4 DNA ligase buffer with 10mM ATP, 2ul 10mM dNTP, 1ul Klenow DNA pol (1u/ul), 1ul T4 DNA pol and 1ul T4 PNK. The reaction was incubated for 30min at 20C. Products were purified in Qiagen PCR purification column in 35ul. 2. Adding over-hang A to the 3’ end in 50ul reaction containing 34ul DNA, 5ul 10X NEB buffer 2, 10ul 1mM dATP and 1ul Klenow exo-. The reaction was incubated for 30min at 37C. Products were purified in Qiagen PCR Mini elute purification column in 14ul. 3. Adapter ligation in 30ul reaction containing 13ul DNA, 15ul 2X T4 DNA ligase buffer, 1ul of 1:200 dilution of Illumina true seq DNA sample prep kit and 1ul quick T4 DNA ligase. The reaction was incubated for 15min at 25C. Products were purified in Qiagen Mini elute PCR purification column in 15ul. 4. Size selection. Ligated products were loaded onto 2% Agarose gel (1XTAE), and run for 1 hour at 120V. DNA was viewed on low-wave trans illuminator and size selected at the 300-400bp range using the Kb+ invitrogen DNA ladder. Products were purified in Qiagen PCR purification column in 25ul. 5. PCR amplification was done in 50ul reaction containing 25ul 2X master mix (2X Phusion HF, FINZYMES), 2ul PCR primers (illumina) and 23ul DNA. PCR program according to the illumina protocol. Products were purified in Qiagen Mini elute PCR purification column in 15ul. PCR products were then run on an Agilent DNA 1000 chip for quantification and submitted for sequencing on the NextSeq 500 illumina platform.

tag: protocol

value: XL-Mnase ChIP

tag: fixation

value: 10 mins RT, 1% formaldehyde

tag: phenotype

value: Wild Type