title: CTRL Liver small fraction Rep 2

city: Philadelphia

first: dario

last: nicetto

state: PA

address: 3400 Civic Center Blvd

department: Cell and Developmental Biology

institute: University of Pennsylvania

description: CTRL_1630_R1_R2_R3_pooled.LargeOverSmall.Unique.scalarNormalizedDivision.bw

library_source: genomic

data_processing:
Alignment: srHC-seq data were aligned to mouse genome assembly mm9/NCBI v37 using bowtie2, version 2.1.0, using the --very-sensitive parameter setting.
Track creation: Read density was visualized using the UCSC Genome Browser to display bigWigs created with BEDTools genomeCoverageBed and UCSC Genome Browser’s bedGraphToBigWig utility with a normalization step that adjusts each track to the library size (RPM) and which takes the fold-change of large/small fractions (fold-change is large/small if large/small > 1; else -small/large)
Genome_build: mm9
Supplementary_files_format_and_content: bigWigs are track files generated in track creation representing the ratio of sonication resistant heterochromatin to open chromatin

source_name: Ctrl 1month old liver

extract_protocol:
We provide a detailed protocol describing how to perform isolation of srHC and how to obtain samples ready to be used for libraries preparation starting from low cell number samples.    A) Related publications: (38–40)    B) Reagents:  • Agencourt® AMPure® XP (Beckman Coulter, cat. no. A63881)   • 80% EtOH  • Buffer EB (10 mM Tris-Cl, pH 8.5)  • Glycine (2M solution) (Fisher BioReagents BP381-1)  • nuclear lysis buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, 1% SDS - Lonza, #51213, supplemented with protease inhibitors)  • TE buffer (10 mM Tris·Cl, pH 8.0, 1 mM EDTA)  • RNase A (Roche, #10109169001)  • Glycogen (Roche, #10901393001)    C) Equipment:  • COVARIS S220 (Focused Ultrasonicator S220)  • microTUBE AFA Fiber Pre-Slit Snap-Cap (COVARIS, cat. #520045)  • DynaMag™-2 Magnet (Thermo Fisher Scientific, #12321D)  • Agilent 2100 Bioanalyzer system (Agilent Technologies, G2939AA)  • Eppendorf Tubes    D) Procedure:  • Cell Preparation  1. Prepare thirty thousand (30,000) cell aliquots in 500 ul PBS from sorted cells.  2. Add 13.5 ul of 37% formaldehyde (Fisher Chemical F79-500) per 500 ul sample.   3. Incubate samples 10 min at RT on rotating platform.  4. Stop crosslinking reaction by adding 36 ul of 2M glycine (Fisher BioReagents BP381-1) to each sample.  5. Incubate samples for 5 min incubation at RT on rotating platform.  6. Centrifuge samples at 2000 RPM for 15 min at 4 °C.  7. Wash cells twice in cold PBS (use 10 ul of FBS - Hyclone, SH30071, in 500 ul of PBS). For each wash, centrifuge samples at 2000 RPM for 5 min at 4 °C. Slowly aspirate supernatant with pipetman, leaving approximately 10 ul of solution behind.  8. Resuspend the cells in 130 ul nuclear lysis buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, 1% SDS - Lonza, #51213, supplemented with protease inhibitors - 1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001-  per 10 ml Buffer).    • Chromatin Sonication  1. Sonicate chromatin using COVARIS S220 following these parameters:   Peak Power  200   Duty Cycle  5%   Burst/Cycle  200   Time   10’    • DNA purification  1. Adjust sample volume to 200ul with nuclear lysis buffer.  2. Reverse crosslinking by incubating samples at 65°C/18hours on heat block`.  3. Add 200ul TE buffer (10 mM Tris·Cl, pH 8.0, 1 mM EDTA), 8uL of 10mg/ml RNase A (Roche, #10109169001) to the samples.   4. Incubate the samples for 2hr at 37 °C.  5. Add 4ul, 20mg/ml Proteinase K (Roche, #03115828001) to the samples.   6. Incubate the samples for 2hr at 55 °C.  7. Purify DNA using conventional phenol:chloroform:isoamyl extraction. Briefly, add 400 ul phenol:chloroform:isoamyl mix to each sample. Mix and quickly vortex the samples.  8. Spin down the samples at max speed for 5 min at RT.  9. Transfer 350 ul of aqueous phase to a new eppendorf  10. Add 16 ul 5M NaCl, 1.5ul 20mg/ml glycogen (Roche, #10901393001) and 800 ul 100% EtOH to each sample.  11. Mix the samples thoroughly by inverting the tube 10-15 times.  12. Incubate the samples are incubated overnight at -80 °C.  13. Centrifuge the samples for 10 min at max speed at 4 °C.  14. Wash the samples with 500 ul 80% EtOH.  15. Centrifuge the samples for 10 min at max speed at 4 °C.  16. Air-dry sample pellet at RT 5 min and resuspend it in 21 ul EB buffer.  17. Save 1 ul purified DNA for checking sonication at the Bioanalyzer (Agilent Technologies, G2939AA) in parallel with further processed samples.    • Sonication-resistant chromatin selection  1. Add 0.7X AMPure XP beads (Beckman Coulter, A63881) to the each sample (14ul in 20ul initial solution). Please note that the amount of AMPure XP beads indicated has been optimized for 3*103 cell samples. Samples of different cell number might require adjustment in AMPure XP beads:volume ratio.  2. Mix samples thoroughly by pipetting 10-12 times.  3. Incubate samples at RT for 5 min.  4. Transfer eppendorf tubes to a magnetic rack (Thermo Fisher Scientific, #12321D) and beads are allowed to separate for 5 min at RT.  5. Transfer clear solution to a new eppendorf tube (this fraction is the small, sonication-sensitive fragments).  6. Wash beads (to which large, sonication-resistant fragments are bound) with 200ul EtOH 80% while tubes are maintained on rack.   7. Incubate samples for 30 sec with EtOH.  8. Remove EtOH and wash once more each sample.  9. Air-dry beads 5 min while tubes are kept on the rack.   10. Remover tubes from the rack  11. Elute the beads in 20 ul TE buffer, by pipetting thoroughly.  12. Save 1 ul purified DNA before further sonication to check samples size at the Bioanalyzer (Agilent Technologies, G2939AA) in parallel with further processed samples.  13. Incubate the samples are incubated 5 min at RT.   14. Place the samples on the magnetic rack (Thermo Fisher Scientific, #12321D).  15. Incubate the samples for 5 min.  16. Transfer clear solution to a new tube (this fraction is the large, sonication-resistant portion of DNA).    • Sonication of large DNA fragments  1. Adjust sample volume to 130ul adding TE buffer.   2. Sonicate the samples using COVARIS S220 (Focused Ultrasonicator S220) following these parameters:  Peak Power  175  Duty Cycle  10%  Burst/Cycle  200  Time   6’    3. Adjust sample volume to 200ul with TE buffer. DNA precipitation can be applied also to small, sonication-sensitive sample.  4. Add 1.0 ul 20mg/ml glycogen (Roche, #10901393001), 1/10 volume of sodium acetate (3 M, pH 5.2) and 3.0X volume EtOH 100% to each sample  5. Mix the samples thoroughly by inverting the tube 10-15 times.  6. Incubate the samples 4h at -80 °C.  7. Centrifuge the samples for 10 min at max speed at 4 °C.  8. Wash the samples with 500 ul 80% EtOH.  9. Centrifuge the samples for 10 min at max speed at 4 °C.  10. Air-dry sample pellet at RT 5 min and resuspend it in 11 ul EB buffer.  11. Save 1 ul of purified DNA for checking sonication at the Bioanalyzer (Agilent Technologies, G2939AA) in parallel with further processed samples.
Libraries were generated using ThruPLEX DNA-seq 48S Kit (Rubicon Genomics, #R400427). Libraries were quantified by qPCR using the NEBNext Library quantification kit for Illumina (NEB, #E7630L). Libraries were diluted to 8 nM and pooled for multiplexing. The diluted concentrations and were quantified a second time using NEBNext kit. Libraries were denatured in 0.2 M NaOH and loaded into NextSeq 75-cycles High Output v2 Kit (Illumina, FC-404-2005) cartridge at a concentration of 2.5-3 pM. Sequencing runs were performed in an Illumina NextSeq 500 machine
srHC-Seq

treatment_protocol: B6C3F1 female were superovulated by intraperitoneal injection (5 IU pregnant mare serum – PMSG, Prospec, cat# hor-272) followed, 48h later, by a second injection (5 IU human chorionic gonadotropin - HCG, Peprotech cat#: 100-39-10UG) and then bred to C3H or Pdx1-eGFP or Ins-RFP males. To generate single cells for FACS, embryos at different developmental stages (i.e. e8.25) were dissected out of decidua and collected in 1.5ml tubes containing 1X PBS on ice. Collected samples were dissociated with 0.05% Trypsin-EDTA (Life Technologies, #25300-054) for 5 min at 37C while gently shaking. The reaction was stopped by adding 0.4 volumes of FBS (Hyclone, SH30071). Explants were homogenized by gentle pipetting. Cells were spun down at 1500rpm for 5min at 4C, and the pellet was resuspended in 320 ul of IMDM (Life Technologies, #12440-053) (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). Twenty (20) ul were transferred into a new tube and 280 ul IMDM were added to it (negative control tube - i.e. samples non incubated with the primary antibody). E8.25 definitive endodermal cells were purified using an antibody detecting ENDM1 (dilution: 1:50) surface antigen (31–33). . Cells were incubated for 30-40 min with primary antibody. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 300 ul of IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). PE-conjugated goat anti rat IgG (Biolegend 405406) was added (dilution 1:300). Samples were mixed well and incubated for 30 min on ice in the dark. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 400-800ul 1XPBS + 3% BSA (Sigma A9418-50G). Samples were filtered through a cell strainer (FALCON 5ml polystyrene round-bottom tube with cell-strainer cap, #352235). Samples were kept on ice until sorting. Mature beta cells were isolated from Ins-RFP transgenic embryos as described (34).Two (2) -month old mice were anesthetized using isoflurane (Butler Animal Health Supply, #029405) and monitored by paw pinch.  A V-shaped incision was used to reveal the abdominal cavity, the intestines moved aside, and a 22 or 24 gauge catheter (Midwest Veterinary Supply, #381423) was used to cannulate the venae cavae while liver perfusion media pumps into the liver, and the portal vein is immediately severed. 37°C liver perfusion media (Invitrogen #17701-038) and then liver digest media (Invitrogen #17703-034) are perfused through the liver (45mL each per animal).  Livers are dissociated with a cutting motion with cell scrapers in William’s E Medium (Sigma W4128) and strained through a 100μm filter (BD 352360).   Hepatocytes are loosely pelleted by centrifugation at 50g for 5 min at 4°C. Twenty-five ul of the hepatocytes pellet were transferred into RLT buffer (RNeasy minikit). One (1) month old control and triple knockout (TKO) mutant liver were dissected and cut in pieces. A small piece was transferred to 1.5 ml tube. Samples were minced using dissecting scissors. One (1) ml ACK (Life Technologies, #A10492-01) solution was added and samples were incubated 5-7 min on ice. Samples were spun down at 1,500 rpm for 5 min at 4C; pellet was resuspended in 500 ul 1XPBS and dounced with microcentrifuge tubes pellet pestle (DWK Life Sciences – Kimble - #7495150000) for 10-15 sec on ice. Samples were spun down at 1,500 rpm for 5 min at 4C.Pellet was resuspended in RLT buffer (RNeasy minikit).31.P. Gadue et al., Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm. Stem Cells Dayt. Ohio. 27, 2103–2113 (2009).32.C.-R. Xu et al., Chromatin “prepattern” and histone modifiers in a fate choice for liver and pancreas. Science. 332, 963–966 (2011).33.C.-R. Xu et al., Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification. EMBO J. 33, 2157–2170 (2014).34.D.-S. Li, Y.-H. Yuan, H.-J. Tu, Q.-L. Liang, L.-J. Dai, A protocol for islet isolation from mouse pancreas. Nat. Protoc. 4, 1649–1652 (2009).

tag: embryonic stage

value: 4 weeks

tag: fragment size cutoff


title: CTRL Liver large fraction Rep 3

status: Public on Oct 31 2018

type: SRA

country: USA

molecule: genomic DNA

organism: Mus musculus


title: CTRL Liver small fraction Rep 1


title: MSA102_G09_trichostatin NA 6_hours 3121-2-1

type: RNA

city: Tempe

first: Ben

last: Readhead

state: AZ

address: 797 E. Tyler Street

institute: ASU-Banner Neurodegenerative Disease Research Center

description: 24 hiPSC-derived NPCs and 8 CCLs were used (total: 32 cell lines). Each 96-well plate included: A1 empty well, 12 vehicle wells, and 2 positive control wells (in duplicate) per cell line (i.e., per plate). The remaining 79 wells were used for test compounds: a) phase 1 had 6/79 drugs from pilot, and 73/79 phase 1 test compounds, b) phase 2 had 6 pilot drugs re-tested, 6/73 phase 1 drugs re-tested, and 67/79 phase 2 compounds. 135 unique compounds were tested.

hyb_protocol: PCR amplicon of each gene is hybridized to barcoded Luminex beads. The barcode incorporated in each amplicon is complementary to a barcode on a Luminex bead, ensuring that each gene from the original lysate mRNA is represented by an amplicon bound to a specific bead.

scan_protocol: Luminex scanners detect individual beads by color, then measure the fluorescent intensity of the SAPE molecules attached to the biotin label on each amplicon-bound bead

data_processing: Raw intensity measurements are deconvoluted to provide intensity values for each landmark gene (the 978 directly measured features). Data is then normalized using a calibration curve based on invariant gene sets, and then quantile normalized.

label: biotin

molecule: total RNA

organism: Homo sapiens

label_protocol: mRNA is reverse transcribed and the cDNA product PCR amplified using primers that contain contiguous sequence (barcode) complementary to each L1000 gene; in addition, the upstream primer is biotinylated to enable identification of amplicon-bound beads.

extract_protocol: Cells were lysed with cell lysis buffer (110 µL per well), and stored at -80⁰C

tag: perturbagen

value: trichostatin

tag: cell id

tag: batch

tag: duration

tag: perturbation type

value: poscon

tag: well id

tag: plate id


title: MSA102_G13_Primidone 10_uM 6_hours 3121-2-1

value: Primidone

tag: dosage

value: test


title: MSA102_G07_emetine 7.2_uM 6_hours 3121-2-1

value: emetine


title: MSA102_G06_melatonin 1_uM 6_hours 2011-3-12

value: melatonin


title: MSA102_G05_melatonin 1_uM 6_hours 3121-2-1


title: MSA102_G11_4-hydroxyphenazone 10_uM 6_hours 3121-2-1


title: MSA102_G04_Loxapine 1_uM 6_hours 2011-3-12

value: Loxapine


title: MSA102_G02_diflorasone 8_uM 6_hours 2011-3-12

value: diflorasone


title: Sample_LI-45

library_source: transcriptomic

data_processing:
Single-end reads were aligned using STAR (Version 2.5.1b)
Mapped reads were counted using subread featurecounts function
Genes with at least 1 count per million mapped reads in at least 5 libraries were retained for further analysis
Gene counts were normalized using the TMM method, and transformed using the voom function
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include a matrix of genes x samples. Values for "Readhead_2018_RNAseq_gene_counts.txt" are gene counts, and values for "Readhead_2018_RNAseq_gene_normalized.txt" are log2 normalized gene abundance.

source_name: SZ_hiPSC NPCs_DMSO

growth_protocol: hiPSC forebrain NPCs were derived as described previously (Brennand et al., 2011 Nature; Hoffman et al, 2017 Nat Communications). For hiPSC NPCs, growth media used was DMEM/F12 + GlutaMAX™ -I (ThermoFisher Scientific, Cat. No. 10565, Lot No. 1715888) containing 1x N2 (ThermoFisher Scientific, Cat. No. 17502-048, Lot No. 1672893), 1x B27-RA (ThermoFisher Scientific, Cat. No. 12587-010, Lot No. 1731195) and 20 ng·ml−1 FGF2 (R&D Systems, Cat. No. 233-FB, Lot No. HKW12815061). Accutase for cell dissociation was from Innovative Cell Technologies (Cat. No. AT-104, Lot No. 5S2415A). Cyto One (USA Scientific) and Corning (Fisher Scientific) culture plasticware was used for expansion and plating of cells. hiPSC NPCs were grown on Matrigel (BD Biosciences, Cat. No. 354230, Lot No. 3018665) coated plates in NPC media. NPC experiments were conducted on myocoplasma-free passage-matched populations, generally between passages five and seven.

extract_protocol: RNA libraries were prepared for sequencing using standard Illumina protocols (Ribo-Zero)

treatment_protocol: NPCs were treated for 6 hours with equivalent volumes of DMSO, methylparaben (10uM) or loxapine (1uM) for 6 hours.

tag: diagnosis

value: SZ

tag: cell type

value: hiPSC Neural Progenitor Cells

tag: treatment

value: DMSO


title: Sample_LI-46

source_name: SZ_hiPSC NPCs_MPB

value: MPB


title: MSA102_G01_diflorasone 8_uM 6_hours 3121-2-1


title: MSA102_G03_Loxapine 1_uM 6_hours 3121-2-1


title: MSA102_F24_LY379268 0.1_uM 6_hours SH-SY5Y

source_name: cancerCells


title: MSA102_F22_methylbenzethonium chloride 8.6_uM 6_hours SH-SY5Y

value: methylbenzethonium chloride


title: CTRL Liver small fraction Rep 3


title: Sample_LI-43


title: Sample_LI-42


title: Sample_LI-41


title: CTRL Liver large fraction Rep 1


title: Sample_LI-40


title: TKO sample 2 Liver small fraction Rep 2

description: TKO_1619_1620_pooled.LargeOverSmall.Unique.scalarNormalizedDivision.bw

source_name: TKO 1month old liver


title: Sample_LI-44


title: TKO sample 1 Liver small fraction Rep 2


title: TKO sample 2 Liver large fraction Rep 1


title: TKO sample 2 Liver large fraction Rep 2


title: Sample_LI-38

source_name: Control_hiPSC NPCs_MPB

value: Control


title: TKO sample 1 Liver small fraction Rep 1


title: MSA102_F23_LY379268 0.1_uM 6_hours MCF7


title: MSA102_F14_diphemanil metilsulfate 10_uM 6_hours SH-SY5Y

value: diphemanil metilsulfate


title: MSA102_F15_tyloxapol 4_uM 6_hours MCF7

value: tyloxapol


title: MSA102_F17_DMSO NA 6_hours MCF7

value: vehicle


title: MSA102_F18_DMSO NA 6_hours SH-SY5Y


title: MSA102_F20_anisomycin 10_uM 6_hours SH-SY5Y

value: anisomycin


title: Sample_LI-39


title: MSA102_F19_anisomycin 10_uM 6_hours MCF7


title: MSA102_F12_DMSO NA 6_hours SH-SY5Y


title: MSA102_F21_methylbenzethonium chloride 8.6_uM 6_hours MCF7


title: TKO sample 1 Liver large fraction Rep 2

value: 4 weeks


title: MSA102_F16_tyloxapol 4_uM 6_hours SH-SY5Y


title: MSA102_F13_diphemanil metilsulfate 10_uM 6_hours MCF7


title: Sample_LI-37

source_name: Control_hiPSC NPCs_DMSO


title: MSA102_F08_bumetanide 10_uM 6_hours SH-SY5Y

value: bumetanide


title: MSA102_F11_DMSO NA 6_hours MCF7


title: MSA102_F10_nordihydroguaiaretic acid 1_uM 6_hours SH-SY5Y

value: nordihydroguaiaretic acid

value: test


title: CTRL Liver large fraction Rep 2


title: TKO sample 1 Liver large fraction Rep 1


title: MSA102_F09_nordihydroguaiaretic acid 1_uM 6_hours MCF7


title: Sample_LI-36


title: Sample_LI-34


title: MSA102_F07_bumetanide 10_uM 6_hours MCF7


title: Sample_LI-33


title: MSA102_F03_nefopam 1.38_uM 6_hours MCF7

value: nefopam


title: Sample_LI-32


title: Hepatocytes small fraction Rep 1

description: HEP__Big_divided_Small_INPUT_merged_rep.bw

source_name: Adult hepatocytes

value: 8 weeks


title: MSA102_F06_furosemide 10_uM 6_hours SH-SY5Y

value: furosemide


title: MSA102_F05_furosemide 10_uM 6_hours MCF7


title: MSA102_F04_nefopam 1.38_uM 6_hours SH-SY5Y


title: Hepatocytes large fraction Rep 2


title: Hepatocytes large fraction Rep 1


title: Sample_LI-31

value: SZ


title: Sample_LI-30

value: hiPSC Neural Progenitor Cells


title: Hepatocytes small fraction Rep 2


title: Definitive endoderm small fraction Rep 2

description: ENDM1_Big_divided_Small_INPUT_merged_rep.bw

source_name: Definitiive endoderm (ENDM1+ FACS)


title: MSA102_F01_phenazone 1_uM 6_hours MCF7

value: phenazone


title: MSA102_E21_methylbenzethonium chloride 8.6_uM 6_hours 3121-2-1


title: Definitive endoderm small fraction Rep 1


title: MSA102_E23_LY379268 0.1_uM 6_hours 3121-2-1


title: MSA102_E24_LY379268 0.1_uM 6_hours 2011-3-12


title: TKO sample 2 Liver small fraction Rep 1


title: MSA102_E19_anisomycin 10_uM 6_hours 3121-2-1


title: MSA102_E22_methylbenzethonium chloride 8.6_uM 6_hours 2011-3-12


title: MSA102_E20_anisomycin 10_uM 6_hours 2011-3-12


title: Sample_LI-29

value: DMSO


title: Sample_LI-28

value: MPB


title: MSA102_E17_DMSO NA 6_hours 3121-2-1


title: MSA102_E18_DMSO NA 6_hours 2011-3-12


title: MSA102_F02_phenazone 1_uM 6_hours SH-SY5Y


title: Definitive endoderm large fraction Rep 2


title: Sample_LI-25

value: Control


title: MSA102_E14_diphemanil metilsulfate 10_uM 6_hours 2011-3-12

value: diphemanil metilsulfate


title: Definitive endoderm large fraction Rep 1


title: Sample_LI-27


title: Sample_LI-24

source_name: SZ_hiPSC NPCs_LOX

value: LOX


title: MSA102_E12_DMSO NA 6_hours 2011-3-12

value: vehicle


title: MSA102_E13_diphemanil metilsulfate 10_uM 6_hours 3121-2-1


title: Mature beta cells small fraction Rep 2

description: 8w_INS_RFP_large_divided_small_fraction_R1R2.bw

source_name: Mature beta cells (RFP+ FACS)


title: Sample_LI-20


title: Mature beta cells large fraction Rep 1


title: MSA102_E16_tyloxapol 4_uM 6_hours 2011-3-12

value: tyloxapol


title: Sample_LI-19


title: Mature beta cells small fraction Rep 1


title: Sample_LI-18


title: TKO liver H3K9me3 ChIP Rep 1

description: TKO_pooled_H3K9me3_spike_in_normalized_track.bw

data_processing:
Alignment: ChIP-seq data were aligned to mouse genome assembly mm9/NCBI v37 using bowtie2, version 2.1.0, using the --very-sensitive parameter setting.
Track creation: Read density was visualized using the UCSC Genome Browser to display bigWigs created with BEDTools genomeCoverageBed and UCSC Genome Browser’s bedGraphToBigWig utility with a normalization step that adjusts each track to the library size (RPM) and which takes the fold-change of H3K9me3/Input or H3K27me3/Input (fold-change is defined as chIP/input if chIP/input > 1, otherwise -input/chIP
H3K27me3 table: area-under-the-curve (AUC) measurements were taken for all gene bodies, summing tag counts and adjusting by the reads per million coefficient (RPM) for chIP and input, and then taking the fold-change and adjusting for gene body length to get a normalized tag density.
Genome_build: mm9
Supplementary_files_format_and_content: bigWigs are UCSC Genome Browser tracks and the text table describes H3K27me3 enrichment over gene bodies

extract_protocol:
B6C3F1 female were superovulated by intraperitoneal injection (5 IU pregnant mare serum – PMSG, Prospec, cat# hor-272) followed, 48h later, by a second injection (5 IU human chorionic gonadotropin - HCG, Peprotech cat#: 100-39-10UG) and then bred to C3H or Pdx1-eGFP or Ins-RFP males. To generate single cells for FACS, embryos at different developmental stages (i.e. e8.25, e10.5, e11.5, e18.5) were dissected out of decidua and collected in 1.5ml tubes containing 1X PBS on ice. For e10.5 and e11.5 embryos, most of the dorsal part (somites) was removed. E18.5 pancreata, clearly visible and easy to distinguish, were dissociated from the rest of the organs. Collected samples were dissociated with 0.05% Trypsin-EDTA (Life Technologies, #25300-054) for 5 min at 37C while gently shaking. The reaction was stopped by adding 0.4 volumes of FBS (Hyclone, SH30071). Explants were homogenized by gentle pipetting. Cells were spun down at 1500rpm for 5min at 4C, and the pellet was resuspended in 320 ul of IMDM (Life Technologies, #12440-053) (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). Twenty (20) ul were transferred into a new tube and 280 ul IMDM were added to it (negative control tube - i.e. samples non incubated with the primary antibody). E8.25 definitive endodermal cells were purified using an antibody detecting ENDM1 (dilution: 1:50) surface antigen (31–33). E10.5 hepatoblasts were sorted from Pdx1-GFP transgenic embryos, staining the cells for the surface marker Liv2 (MBL, cat# D118-3, dilution: 1:100). At the same stage pancreatic precursor Pdx1-GFP positive cells were collected. A subpopulation of Liv2+/Pdx1-GFP+ cells was detected at E10.5, as previously reported (33). These cells were not considered for further analysis. E11.5 hepatoblasts were sorted from wt embryos using the same antibody and conditions as for e10.5 embryos. Cells were incubated for 30-40 min with primary antibody. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 300 ul of IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). PE-conjugated goat anti rat IgG (Biolegend 405406) was added (dilution 1:300). Samples were mixed well and incubated for 30 min on ice in the dark. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 400-800ul 1XPBS + 3% BSA (Sigma A9418-50G). Samples were filtered through a cell strainer (FALCON 5ml polystyrene round-bottom tube with cell-strainer cap, #352235). Samples were kept on ice until sorting. Mature beta cells were isolated from Ins-RFP transgenic embryos as described (34).Two (2) -month old mice were anesthetized using isoflurane (Butler Animal Health Supply, #029405) and monitored by paw pinch.  A V-shaped incision was used to reveal the abdominal cavity, the intestines moved aside, and a 22 or 24 gauge catheter (Midwest Veterinary Supply, #381423) was used to cannulate the venae cavae while liver perfusion media pumps into the liver, and the portal vein is immediately severed. 37°C liver perfusion media (Invitrogen #17701-038) and then liver digest media (Invitrogen #17703-034) are perfused through the liver (45mL each per animal).  Livers are dissociated with a cutting motion with cell scrapers in William’s E Medium (Sigma W4128) and strained through a 100μm filter (BD 352360).   Hepatocytes are loosely pelleted by centrifugation at 50g for 5 min at 4°C. Twenty-five ul of the hepatocytes pellet were transferred into RLT buffer (RNeasy minikit). One (1) month old control and triple knockout (TKO) mutant liver were dissected and cut in pieces. A small piece was transferred to 1.5 ml tube. Samples were minced using dissecting scissors. One (1) ml ACK (Life Technologies, #A10492-01) solution was added and samples were incubated 5-7 min on ice. Samples were spun down at 1,500 rpm for 5 min at 4C; pellet was resuspended in 500 ul 1XPBS and dounced with microcentrifuge tubes pellet pestle (DWK Life Sciences – Kimble - #7495150000) for 10-15 sec on ice. Samples were spun down at 1,500 rpm for 5 min at 4C.Pellet was resuspended in RLT buffer (RNeasy minikit).31.P. Gadue et al., Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm. Stem Cells Dayt. Ohio. 27, 2103–2113 (2009).32.C.-R. Xu et al., Chromatin “prepattern” and histone modifiers in a fate choice for liver and pancreas. Science. 332, 963–966 (2011).33.C.-R. Xu et al., Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification. EMBO J. 33, 2157–2170 (2014).34.D.-S. Li, Y.-H. Yuan, H.-J. Tu, Q.-L. Liang, L.-J. Dai, A protocol for islet isolation from mouse pancreas. Nat. Protoc. 4, 1649–1652 (2009).
We provide here a detailed protocol describing how to prepare low cell number sample for chromatin-related experiments.  A) Notes:  The following protocol is modified from O’Geen, et al. (37) and recommended for small cell number ChIP-seq and is based on successful 3 x 10^4 cells for ChIP-seq with H3K9me3 and H3K27me3 antibodies in mouse sorted cells isolated by FACS with cell surface markers at different developmental stages (i.e. e8.25, e10.5). Please note for optimal DNA recovery when purifying ChIP, use a Phenol Chloroform method. This protocol was chosen due to its high specificity and low background.     B) Buffers:  Cell Lysis Buffer (make 10 ml fresh each time)  3mM MgCl2  10mM NaCl  10mM Tris, pH 7.4  0.1% Igepal (Sigma Aldrich, cat #I8896)  Add protease inhibitors (1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001-, per 10 ml Buffer).     Nuclei Lysis Buffer (prepare it fresh each time)   50mM Tris-Cl pH 8.0  10mM EDTA  1% SDS (Lonza, #51213)  Before use, add protease inhibitors (1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001- per 10 ml Buffer. Never store at 4C).    IP Dilution Buffer (RIPA)   50mM Tris pH 7.4  150mM NaCl  1% Igepal (Sigma Aldrich, cat #I8896)  0.25% Sodium Deoxycholate (Sigma Aldrich, cat #D6750)  1mM EDTA  Add protease inhibitors (1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001- per 10 ml Buffer).    IP Wash Buffer 1 (RIPA without protease inhibitors; place it on ice after preparation)  50mM Tris pH 7.4  150mM NaCl  1% Igepal (Sigma Aldrich, cat #I8896)  0.25% Deoxycholic Acid (Sigma Aldrich, cat #D6750)  1mM EDTA    IP Wash Buffer 2 (place it on ice after preparation)  100mM Tris-Cl pH 9.0  500mM LiCl  1% Igepal (Sigma Aldrich, cat #I8896)  1% Deoxycholic acid (Sigma Aldrich, cat #D6750)    IP Wash Buffer 3 (IP Wash Buffer 2 plus NaCl; place it on ice after preparation)  100mM Tris-Cl pH 9.0  500mM LiCl  1% Igepal (Sigma Aldrich, cat #I8896)  1% Deoxycholic acid (Sigma Aldrich, cat #D6750)  150mM NaCl    Elution Buffer   50mM NaHCO3  1% SDS (Lonza, #51213)    C) Cell crosslinking:  The following has been optimized for crosslinking sorted cells:  1. Use FACs to sort > 104 cells into 1.5 ml tube, containing 50 ul 1X PBS supplemented with 1% BSA (Sigma A9418-50G) and 20% FBS  (Hyclone, SH30071). Spin down the cells at 1700rpm for 12-15 min at 4C. Resuspend the pellet in 200-300ul PBS freshly prepared. Prepare 3 x 104 cells aliquots in 500ul PBS or alternatively, if you have a lot of cells, prepare 1-3 x 105 cells aliquots.  2. Add 13.5 ul of 37% formaldehyde (Fisher Chemical F79-500) per 500 ul sample. Incubate exactly 10 min at room temp on rotating platform.  3. Stop crosslinking reaction by adding 36 ul of 2M glycine (Fisher BioReagents BP381-1) to the sample. Incubate 5 min at room temp on rotating platform.  4. Centrifuge at 2000 RPM for 15 min at 4C  5. Wash cells twice in cold PBS supplemented with 2% FBS  (Hyclone, SH30071). For each wash, centrifuge at 2000 RPM for 5 min. Slowly aspirate supernatant with pipetman, leaving approximately 10 ul of solution behind. Be careful to not disturb the pellet.       D) Preparation of chromatin from crosslinked cells:  1. If you have > 105 cells, resuspend cells in 300 ul freshly prepared cell lysis buffer and incubate on ice for 15 min. Centrifuge at 5000 RPM for 5 min at 4 C. Repeat centrifugation if cell lysate is not visible. Remove supernatant with pipetman, leaving pellet. Leave approximately 10 ul of supernatant. If starting with < 105 cells, proceed to step 2.  2. Prepare aliquots of 3 x 104 cells in 130 ul Nuclei Lysis Buffer.  3. Incubate on ice for 15 minutes, flash freeze (use dry ice or liquid nitrogen), then thaw to room temperature to aid in lysis. (You may keep chromatin at -80 C until a future day and then thaw to room temperature).  4. Sonicate with predetermined conditions so that chromatin is between 200 and 500 bp in length. If using Covaris (S220 Focused Ultrasonicator) for chromatin sonication apply the following parameters:  Incident peak power: 200  Duty Cycle: 5%  Burst/Cycle: 200  Time: 10-12min  5. Centrifuge sonicated chromatin for 10 min at 14K RPM at 4C, and collect supernatant and either use immediately in ChIP experiment or freeze at -80C.    E) Checking sonicated chromatin:  1. Collect about 3*103 cells and resuspend them in elution buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS - Lonza, #51213)  2. Reverse crosslink at 65 C o/n on heat block  3. Add 200 ul TE buffer (to dilute SDS - Lonza, #51213)  4. Add 8 ul of 10 mg/ml RNase A (Roche, #10109169001) and incubate samples for 1.5-2 h at 37 C  5. Add 4ul Proteinase K (20mg/ml stock) (Roche, #03115828001) and incubate 1.5-2 h at 55C  6. Precipitate DNA with standard phenol-chloroform protocol  7. Transfer the aqueous phase to a new tube  8. Add 16 ul of 5 M NaCl, 1.5 ul of 20 mg/ml glycogen (Roche, #10901393001) and 800 ul 100% EtOH  9. Place the samples at -80 C for a couple of hours (you can do this step o/n)  10. Pellet DNA spinning the samples at 15,000 rpm for 12 min at 4 C (pellet is faint!)  11. Wash with 500 ul 80% EtOH, once  12. Pellet DNA spinning the samples at 15,000 rpm for 12 min at 4 C  13. Air dry the pellet and resuspend in 15 ul TE buffer (remove EtOH)  14. Spin vacuum the samples and resuspend them in 3ul H2O containing 10% glycerol  15. Prepare 1% agarose gel (as thin as possible) without Ethidium Bromide  16. Load the samples and marker   17. Run the gel at 90V for 40-45 min  18. Stain the gel (10 ug/ml final concentration) for 15 min RT while shaking  19. Wash the gel 5 min RT while shaking in ddH2O  20. Image the gel.  Note: the sonication can be checked with the Bioanalyzer (Agilent Technologies G2939AA) by resuspending the samples from step 14 in water (3 ul).    Chromatin Immunoprecipitation (ChIP) on low cell number samples:  We provide here a detailed protocol describing how to perform chromatin immunoprecipitation and how to obtain samples ready to be used for libraries preparation starting from low cell number samples.    A) Immunoprecipitation  1. Transfer sonicated chromatin into 0.6 ml Axygen Maxymum Recovery microtubes (MCT-060-L-C, #311-03-051). Dilute chromatin with Dilution Buffer to a final volume of 500 ul. For input, take 5 ul (1%) from the 500ul containing sheared chromatin and transfer to new tube containing 100 ul elution buffer.  2. Add 1 ug of primary ChIP-grade antibody and incubate overnight on a rotating platform at 4 deg.  3. Add 15 ul Cell Signaling ChIP-grade Protein G magnetic Beads (Invitrogen #10004D) and incubate 2 hours on rotating platform at 4 deg.  4. Transfer chromatin and beads to a pre-chilled 1.5 ml microtubes and allow beads to separate in magnetic rack (Thermo Fisher Scientific, #12321D) for > 30 seconds.  5. Remove supernatant with pipetman. Using suction may cause you to lose beads and sample.   6. Remove tube from rack, resuspend beads in IP Wash Buffer 1, and wash by pipetting up and down 12 times.  7. Place tubes in magnetic rack  (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.  8. Repeat steps 5-7 once.  9. Remove supernatant with pipetman.  10. Remove tubes from rack, resuspend beads in IP Wash Buffer 2, and wash by pipetting up and down 12 times.  11.  Place tubes in magnetic rack (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.  12. Repeat steps 9-11 2 times.  13.  Remove supernatant with pipetman.  14.  Remove tubes from rack, resuspend beads in IP Wash Buffer 3, and wash by pipetting up and down 12 times.  15.  Transfer beads and chromatin to a new 1.5 ml microtube.  16.  Place in magnetic rack (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.  17.  Remove tubes from rack and resuspend in 100 ul Elution Buffer.  18.  Shake at RT at a setting of 1300 RPM for 1 hour.  19.  Place tubes in magnetic rack (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.   20.  Collect supernatant and add 12 ul 5M NaCl.  21. Thaw the input and add 12ul 5M NaCl  22.  Incubate 4 hours to overnight at 65 deg.  23.  Add 3 ul of 10 mg/ml RNase A (Roche, #10109169001) to each sample and incubate at 37 C for 30 min.    B) Phenol Chloroform sample purification  1. Spin down Phase Lock Gel Light 1.5 ml tube (5 Prime, QuantaBio, #2302820) at 14000 rpm for 30sec.  2. Add 100ul TE buffer to each samples (including input) and mix gently (total volume 200ul)  3. Mix 200 ul ChIP product and 200 ul Phenol/Chloroform/Isoamyl Alcohol by pipetting about 10 times and add to Phase Lock Gel Light 1.5 ml tube. Do not vortex.  4. Centrifuge at room temperature for 5 minutes at 14000 RPM.  5. Optional: Add an additional 200 ul of Phenol/Chloroform/Isoamyl Alcohol and mix by pipetting.  6. Centrifuge at room temperature for 5 minutes at 14000 RPM.  7. Collect aqueous solution on top and add to a new tube (should be about 200 ul). If the volume is less than 200 ul, add Buffer TE to reach 200 ul.   8. Add 8 ul 5M NaCl, 10 ug glycogen, and 400 ul cold 100% ethanol, and mix.  9. Precipitate DNA in ethanol at -80 overnight at least for best recovery.   10. Centrifuge at 4 C for 10 min at 11000 RPM.  11. Aspirate supernatant carefully, without disturbing the very small white pellet. To be safe, you may leave 5 ul at the bottom.  12.  Wash with 500 ul 80% ethanol by pipetting five times.  13.  Centrifuge at 4 C for 10 min at 11000 RPM.  14.  Aspirate supernatant carefully, without disturbing the very small white pellet.   15.  Air dry 5 minutes, or until ethanol has evaporated, but pellet isn’t completely dry. If pellet is too dry, it will be difficult to resuspend.  16. Resuspend pellet in 30-40 ul Qiagen EB. It is now ready for qPCR. For library preparation resuspend the pellet in less volume (e.g. 10ul).
Libraries were generated using ThruPLEX DNA-seq 48S Kit (Rubicon Genomics, #R400427). Libraries were quantified by qPCR using the NEBNext Library quantification kit for Illumina (NEB, #E7630L). Libraries were diluted to 8 nM and pooled for multiplexing. The diluted concentrations and were quantified a second time using NEBNext kit. Libraries were denatured in 0.2 M NaOH and loaded into NextSeq 75-cycles High Output v2 Kit (Illumina, FC-404-2005) cartridge at a concentration of 2.5-3 pM. Sequencing runs were performed in an Illumina NextSeq 500 machine

value: 4 weeks

tag: antibody

value: Abcam ab8898


title: MSA102_E11_DMSO NA 6_hours 3121-2-1


title: Sample_LI-17


title: MSA102_E08_bumetanide 10_uM 6_hours 2011-3-12

value: bumetanide


title: MSA102_E10_nordihydroguaiaretic acid 1_uM 6_hours 2011-3-12

value: nordihydroguaiaretic acid

value: test


title: MSA102_E09_nordihydroguaiaretic acid 1_uM 6_hours 3121-2-1


title: Mature beta cells large fraction Rep 2

value: 8 weeks


title: MSA102_E06_furosemide 10_uM 6_hours 2011-3-12

value: furosemide


title: MSA102_E05_furosemide 10_uM 6_hours 3121-2-1


title: MSA102_E02_phenazone 1_uM 6_hours 2011-3-12


title: TKO liver H3K9me3 ChIP Rep 2


title: MSA102_E04_nefopam 1.38_uM 6_hours 2011-3-12

value: nefopam


title: MSA102_E07_bumetanide 10_uM 6_hours 3121-2-1


title: TKO liver input for H3K9me3


title: MSA102_E01_phenazone 1_uM 6_hours 3121-2-1


title: MSA102_D24_quinpirole 0.1_uM 6_hours SH-SY5Y

value: quinpirole


title: MSA102_E03_nefopam 1.38_uM 6_hours 3121-2-1


title: CTRL liver H3K9me3 ChIP Rep 2

description: 1630_CTRL_pooled_H3K9me3_spike_in_normalized_track.bw


title: MSA102_D23_quinpirole 0.1_uM 6_hours MCF7


title: Sample_LI-16

source_name: Control_hiPSC NPCs_LOX

value: hiPSC Neural Progenitor Cells


title: Sample_LI-23

value: SZ

value: DMSO


title: Sample_LI-14

value: Control


title: CTRL input for H3K9me3 Rep 2


title: Sample_LI-15


title: MSA102_D20_haloperidol 0.1_uM 6_hours SH-SY5Y

value: haloperidol


title: Sample_LI-13


title: CTRL liver H3K9me3 ChIP Rep 1


title: MSA102_D22_hydroquinine 1_uM 6_hours SH-SY5Y

value: hydroquinine


title: Mat. Beta cells H3K27me3 ChIP Rep 2

description: H3K27me3-Input Quantile-Normalized.xlsx

value: Millipor 07-449


title: MSA102_D21_hydroquinine 1_uM 6_hours MCF7


title: MSA102_D19_haloperidol 0.1_uM 6_hours MCF7


title: Sample_LI-10

value: LOX


title: Mat. Beta cells H3K27me3 ChIP Rep 1

description: H3K27me3-Input Quantile-Normalized.xlsx


title: Sample_LI-11


title: Sample_LI-12


title: CTRL liver input for H3K9me3 Rep 1

value: 4 weeks


title: Mat. Beta cells Input for H3K72me3 Rep 2

description: H3K27me3-Input Quantile-Normalized.xlsx


title: MSA102_D17_ethionamide 10_uM 6_hours MCF7

value: ethionamide


title: MSA102_D18_ethionamide 10_uM 6_hours SH-SY5Y


title: Mat. Beta cells Input for H3K72me3 Rep 1

description: H3K27me3-Input Quantile-Normalized.xlsx


title: MSA102_D06_DMSO NA 6_hours SH-SY5Y

value: vehicle


title: MSA102_D13_tanespimycin NA 6_hours MCF7

value: tanespimycin

value: poscon


title: MSA102_D16_zardaverine 10_uM 6_hours SH-SY5Y

value: zardaverine

value: test


title: Hepatocytes H3K27me3 ChIP Rep 2

description: H3K27me3-Input Quantile-Normalized.xlsx

value: 8 weeks


title: MSA102_D07_Vorinostat 1_uM 6_hours MCF7

value: Vorinostat


title: MSA102_D10_DMSO NA 6_hours SH-SY5Y


title: MSA102_D15_zardaverine 10_uM 6_hours MCF7


title: MSA102_D14_tanespimycin NA 6_hours SH-SY5Y


title: MSA102_D11_BAS-012416453 10_uM 6_hours MCF7


title: MSA102_D04_alpha-ergocryptine 7_uM 6_hours SH-SY5Y

value: alpha-ergocryptine


title: MSA102_D05_DMSO NA 6_hours MCF7


title: MSA102_D12_BAS-012416453 10_uM 6_hours SH-SY5Y


title: Sample_LI-09

value: hiPSC Neural Progenitor Cells


title: MSA102_D08_Vorinostat 1_uM 6_hours SH-SY5Y


title: MSA102_D09_DMSO NA 6_hours MCF7

value: DMSO


title: Sample_LI-06

value: Control


title: MSA102_C23_quinpirole 0.1_uM 6_hours 3121-2-1

value: quinpirole


title: MSA102_D02_moxonidine 1_uM 6_hours SH-SY5Y

value: moxonidine


title: Sample_LI-07


title: Sample_LI-08


title: Hepatocytes H3K27me3 ChIP Rep 1

description: H3K27me3-Input Quantile-Normalized.xlsx

value: Millipor 07-449


title: Hepatocytes Input for H3K27me3 Rep 1

description: H3K27me3-Input Quantile-Normalized.xlsx


title: Definitive Endoderm H3K27me3 ChIP Rep 2

description: H3K27me3-Input Quantile-Normalized.xlsx


title: Sample_LI-04

value: SZ

value: LOX


title: Definitive Endoderm H3K27me3 ChIP Rep 1

description: H3K27me3-Input Quantile-Normalized.xlsx


title: Hepatocytes Input for H3K27me3 Rep 2

description: H3K27me3-Input Quantile-Normalized.xlsx


title: Definitive Endoderm input for H3K27me3 Rep 2

description: H3K27me3-Input Quantile-Normalized.xlsx


title: Sample_LI-02


title: MSA102_D01_moxonidine 1_uM 6_hours MCF7


title: MSA102_D03_alpha-ergocryptine 7_uM 6_hours MCF7


title: Sample_LI-03


title: Sample_LI-05


title: Sample_LI-01


title: MSA102_C24_quinpirole 0.1_uM 6_hours 2011-3-12


title: Definitive Endoderm input for H3K27me3 Rep 1

description: H3K27me3-Input Quantile-Normalized.xlsx


title: MSA102_C21_hydroquinine 1_uM 6_hours 3121-2-1

value: hydroquinine


title: VO_pDAPT_P-65967

city: Vienna

first: Reiner

last: Wimmer

address: Dr. Bohrgasse 3

country: Austria

institute: IMBA

data_processing:
Reads mapping to human rRNA transcripts were removed using bwa/0.7.12 alignment and samtools/1.3.1. Remaining reads were aligned to human genome hg38 using TopHat v2.1.1 (GTF annotation file hg38, RefSeq from UCSC, 2015/01; library-type fr-unstranded). Reads in genes were counted with htseq-count v0.6.1.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files with per gene read counts

source_name: iPSC derived

extract_protocol: cDNA synthesis and amplification were performed from total RNA using SMARTer RNA Kit (Clontech) according to the manufacturers instructions.

treatment_protocol: Vascular organoids were cultured in either control condition (17mM Glucose), diabetic condition (75mM Glucose + 1ng/mL TNFalpha +1ng/mL Il6) or pDAPT condition (=diabetic + 25mM DAPT) for 2weeks. Endothelial cells and Pericytes were sorted by FACS and subjected to RNA equencing.

tag: cell line

value: iPSC line NC8

value: pericyte (differentiated from iPS cells)

tag: gender

value: male

tag: culture protocol

value: pDAPT


title: MSA102_C18_ethionamide 10_uM 6_hours 2011-3-12

value: ethionamide

value: test


title: MSA102_C20_haloperidol 0.1_uM 6_hours 2011-3-12

value: haloperidol


title: VO_pDAPT_P-65964


title: MSA102_C19_haloperidol 0.1_uM 6_hours 3121-2-1


title: MSA102_C17_ethionamide 10_uM 6_hours 3121-2-1


title: Midbrain neuroepith. Input for H3K9me3 Rep 2

source_name: Midbrain dissected

tag: embryonic stage


title: VO_pDAPT_P-65966


title: Midbrain neuroepith. Input for H3K9me3 Rep 1


title: Midbrain neuroepith. H3K9me3 ChIP Rep 2

value: Abcam ab8898


title: Midbrain neuroepith. H3K9me3 ChIP Rep 1


title: MSA102_C12_BAS-012416453 10_uM 6_hours 2011-3-12


title: MSA102_C13_tanespimycin NA 6_hours 3121-2-1

value: tanespimycin

value: poscon


title: MSA102_C14_tanespimycin NA 6_hours 2011-3-12


title: MSA102_C09_DMSO NA 6_hours 3121-2-1

value: DMSO

value: vehicle


title: MSA102_C10_DMSO NA 6_hours 2011-3-12


title: VO_pDAPT_EC-65961

value: endothelial cell (differentiated from iPS cells)


title: Meso. Prog. H3K9me3 ChIP Rep 2

description: FLK1_H3K9me3_divided_input_May17_R1R2.bw

source_name: Mesoderm progenitors (FLK1+ FACS)


title: VO_pDAPT_EC-65959


title: MSA102_C15_zardaverine 10_uM 6_hours 3121-2-1

value: zardaverine


title: MSA102_C06_DMSO NA 6_hours 2011-3-12


title: MSA102_C07_Vorinostat 1_uM 6_hours 3121-2-1

value: Vorinostat


title: MSA102_C08_Vorinostat 1_uM 6_hours 2011-3-12


title: MSA102_C16_zardaverine 10_uM 6_hours 2011-3-12


title: MSA102_C11_BAS-012416453 10_uM 6_hours 3121-2-1


title: Meso. Prog. H3K9me3 ChIP Rep 1


title: MSA102_C05_DMSO NA 6_hours 3121-2-1


title: MSA102_C04_alpha-ergocryptine 7_uM 6_hours 2011-3-12

value: alpha-ergocryptine


title: Meso. Prog. Input for H3K9me3 Rep 1


title: MSA102_C03_alpha-ergocryptine 7_uM 6_hours 3121-2-1


title: VO_pDAPT_EC-65958


title: VO_Dia_P-65949

value: Dia


title: MSA102_C01_moxonidine 1_uM 6_hours 3121-2-1

value: moxonidine


title: Mat. Beta cells Input for H3K9me3 Rep 1

value: 8 weeks


title: VO_Dia_P-65948

value: iPSC line NC8

value: pericyte (differentiated from iPS cells)

value: male


title: VO_Dia_EC-65947


title: Imm. Beta cells H3K9me3 ChIP Rep 2

source_name: Immature beta cells (RFP+ FACS)


title: Mat. Beta cells H3K9me3 ChIP Rep 2


title: MSA102_B24_trimethadione 10_uM 6_hours SH-SY5Y

value: trimethadione

value: test


title: VO_Dia_EC-65946


title: MSA102_B23_trimethadione 10_uM 6_hours MCF7


title: VO_Dia_EC-65944


title: Imm. Beta cells H3K9me3 ChIP Rep 1

value: Abcam ab8898


title: MSA102_B21_DMSO NA 6_hours MCF7


title: Mat. Beta cells H3K9me3 ChIP Rep 1


title: MSA102_B19_phenelzine 10_uM 6_hours MCF7

value: phenelzine


title: VO_Ctrl_P-65937

value: Ctrl


title: MSA102_B18_naringenin 10_uM 6_hours SH-SY5Y

value: naringenin


title: MSA102_B17_naringenin 10_uM 6_hours MCF7


title: MSA102_B20_phenelzine 10_uM 6_hours SH-SY5Y


title: MSA102_B22_DMSO NA 6_hours SH-SY5Y

value: DMSO

value: vehicle


title: MSA102_B16_Aripiprazole 1_uM 6_hours SH-SY5Y

value: Aripiprazole


title: MSA102_B14_chenodeoxycholic acid 10_uM 6_hours SH-SY5Y

value: chenodeoxycholic acid


title: MSA102_B12_fendiline 10_uM 6_hours SH-SY5Y

value: fendiline


title: MSA102_C02_moxonidine 1_uM 6_hours 2011-3-12


title: MSA102_B15_Aripiprazole 1_uM 6_hours MCF7


title: MSA102_B13_chenodeoxycholic acid 10_uM 6_hours MCF7


title: VO_Ctrl_P-65936


title: MSA102_B11_fendiline 10_uM 6_hours MCF7


title: MSA102_B10_diclofenamide 10_uM 6_hours SH-SY5Y

value: diclofenamide


title: Imm. Beta cells Input for H3K9me3 Rep 1


title: VO_Ctrl_P-65935


title: VO_Ctrl_EC-65932

value: endothelial cell (differentiated from iPS cells)


title: Hepatocytes H3K9me3 ChIP Rep 2

value: 8 weeks


title: MSA102_B07_arachidonyltrifluoromethane 10_uM 6_hours MCF7

value: arachidonyltrifluoromethane


title: MSA102_B09_diclofenamide 10_uM 6_hours MCF7


title: Pancreatic Precursor Input for H3K9me3 Rep 1

source_name: Pancreatic precursor (PDX1-GFP+ FACS)


title: Pancreatic Precursor H3K9me3 ChIP Rep 2


title: Pancreatic Precursor H3K9me3 ChIP Rep 1


title: VO_Ctrl_EC-65928

value: iPSC line NC8


title: Hepatocytes H3K9me3 ChIP Rep 1


title: Placenta_P-66003

source_name: Placenta

value: pericyte (primary cell)

value: unknown


title: Hepatoblasts H3K9me3 ChIP Rep 2

description: H3K9me3-I3_jan17_LIV2_R1R2_merged_rep_Feb17.bw

source_name: Hepatoblasts (FACS Liv2+ FACS)


title: VO_Ctrl_EC-65931

value: male


title: MSA102_A24_trimethadione 10_uM 6_hours 2011-3-12

value: trimethadione

value: test


title: VO_Dia_P-65996

value: pericyte (differentiated from iPS cells)

value: Dia


title: MSA102_A22_DMSO NA 6_hours 2011-3-12

value: DMSO


title: MSA102_B03_dihydroergocristine 1_uM 6_hours MCF7

value: dihydroergocristine


title: MSA102_B05_Chlorpromazine 0.1_uM 6_hours MCF7

value: Chlorpromazine


title: MSA102_A19_phenelzine 10_uM 6_hours 3121-2-1

value: phenelzine


title: Placenta_P-66002

value: Ctrl


title: MSA102_B06_Chlorpromazine 0.1_uM 6_hours SH-SY5Y


title: Hepatocytes Input for H3K9me3 Rep 1


title: MSA102_A21_DMSO NA 6_hours 3121-2-1

value: vehicle


title: MSA102_A23_trimethadione 10_uM 6_hours 3121-2-1


title: MSA102_C22_hydroquinine 1_uM 6_hours 2011-3-12

value: hydroquinine


title: MSA102_A20_phenelzine 10_uM 6_hours 2011-3-12


title: Definitive Endoderm H3K9me3 ChIP Rep 4

value: Abcam ab8898


title: MSA102_B08_arachidonyltrifluoromethane 10_uM 6_hours SH-SY5Y

value: arachidonyltrifluoromethane


title: Placenta_P-66005


title: Patsch_P-66012

value: iPSC line NC8


title: MSA102_A18_naringenin 10_uM 6_hours 2011-3-12

value: naringenin


title: Hepatoblasts H3K9me3 ChIP Rep 1


title: Hepatoblasts Input for H3K9me3 Rep 1


title: Definitive Endoderm H3K9me3 ChIP Rep 5


title: MSA102_B04_dihydroergocristine 1_uM 6_hours SH-SY5Y


title: Patsch_P-66014


title: Patsch_EC-66011

value: endothelial cell (differentiated from iPS cells)


title: MSA102_A16_Aripiprazole 1_uM 6_hours 2011-3-12

value: Aripiprazole


title: Definitive Endoderm H3K9me3 ChIP Rep 1


title: MSA102_A13_chenodeoxycholic acid 10_uM 6_hours 3121-2-1

value: chenodeoxycholic acid


title: Definitive Endoderm H3K9me3 ChIP Rep 2


title: MSA102_A12_fendiline 10_uM 6_hours 2011-3-12

value: fendiline

value: test


title: MSA102_A17_naringenin 10_uM 6_hours 3121-2-1


title: Definitive Endoderm input for H3K9me3 Rep 2


title: MSA102_A11_fendiline 10_uM 6_hours 3121-2-1


title: MSA102_A15_Aripiprazole 1_uM 6_hours 3121-2-1


title: Patsch_P-66015

value: pericyte (differentiated from iPS cells)

value: male


title: NC8_iPSC-66019

source_name: iPSC

value: iPS cell


title: Patsch_EC-66010


title: TKO Liver Rep 2

data_processing:
Alignment: RNA-seq data were aligned to mouse genome build mm9 using STAR version 2.5.2 and default parameters.
Quantitation: Read counts per gene were obtained with HTSeq version 0.6.1.
Differential expression:  Gene counts were analyzed for significant differential expression using DESeq (standard binomial test with an FDR control of 0.1).
Track creation: Genome Browser tracks (bigWig files) were created by pooling both replicates for all samples except ENDM1+ in the developmental trajectory (additionally, the control and triple mutant samples were also unpooled), then using BEDTools genomeCoverageBED to make bedGraph representations of tag density, which were RPM-adjusted; finally, UCSC Genome Browser utility bedGraphToBigWig was used to create bigWig track files.
Genome_build: mm9
Supplementary_files_format_and_content: bigWigs represent track files

molecule: polyA RNA

extract_protocol:
Two replicates were used for each stage. Total RNA was isolated from 3*104 cell aliquots using RNeasy Micro Kit (Qiagen, ID:74004) following manufacturers’ instruction. Total RNA was resuspended in 50 ul BTE solution (10 mM Bis-tris pH6.7, 1 mM EDTA). RNA secondary structures were removed by heating the samples at 65 C for 5 min, followed by incubation on ice. Oligo-dT25 dynabeads (Invitrogen #610-02) were used for selecting polyA mRNA. Beads were washed 3 times with 2X Oligo-dT binding buffer (OBB: 20 mM Tris ph7.5, 1 M LiCl, 2 mM EDTA), resuspended in 50 ul 2X OBB and added to RNA. Samples were incubated 5-10 min at RT while gently shaking. Samples were transferred to a  (Thermo Fisher Scientific, #12321D) rack, and beads were allowed to separate for 3-5 min. Beads were washed 3 times with 200 ul Oligo-dT washing buffer (OWB: 10 mM Tris Ph7.5, 150 mM LiCl, 1 mM EDTA) and polyA mRNA was eluted into 10 ul BTE. Samples were incubated 2 min at 80C and beads were allowed to separate on the magnetic rack (Thermo Fisher Scientific, #12321D). The solution was transferred to a fresh tube. Chemical RNA fragmentation was achieved using Mg2+. On ice, for each 10 ul sample volume, 4 ul 5X 1st strand buffer from SuperScript III RT kit (Invitrogen #18080-044) and 2 ul 50 uM random hexamers (Roche) were added. Samples were incubated 16 min at 94C in PCR machine and ramped down to 4C. First strand synthesis was performed using SuperScript III (Invitrogen #18080-044), following manufacturers’’ instructions and adding 1ug actinomycin D (Sigma #A1410) per reaction. Samples were incubated in PCR machine according to the following scheme: 10 min at 25C, 20 min at 42C, 30 min at 50C, 10 min at 55C. Samples were kept on ice. First strand cDNA was purified using conventional phenol:chloroform:isoamyl extraction. cDNA was precipitated using 3 M NaAc, 1.ul 20mg/ml glycogen (Roche, #10901393001) and  1 ml 100% EtOH. Samples were resuspended in milliQ water. Second strand synthesis incorporating dUTP was performed as following: for each sample, 2 ul First Strand bugger (from SuperScript III kit; Invitrogen #18080-044), 0.5 ul 0.1 M DTT, 5 ul 10X second strand buffer (dNTPs free; NEBnext #B6117S), 2 ul 10 mM dNTPs (with dUPT substituting in place of dTTP), 2 ul DNApol I (20 U; NEB #M0209), 0.5 ul RNAseH (2.5 U: NEB #M0297) were added. Samples were incubated 2-3h at 16C. cDNA was purified using MiniElute kit (Qiagen, #28004) and samples were eluted in 10ul RNase-free water.
Libraries were generated using ThruPLEX DNA-seq 48S Kit (Rubicon Genomics, #R400427). Libraries were quantified by qPCR using the NEBNext Library quantification kit for Illumina (NEB, #E7630L). Libraries were diluted to 8 nM and pooled for multiplexing. The diluted concentrations and were quantified a second time using NEBNext kit. Libraries were denatured in 0.2 M NaOH and loaded into NextSeq 75-cycles High Output v2 Kit (Illumina, FC-404-2005) cartridge at a concentration of 2.5-3 pM. Sequencing runs were performed in an Illumina NextSeq 500 machine

treatment_protocol: B6C3F1 female were superovulated by intraperitoneal injection (5 IU pregnant mare serum – PMSG, Prospec, cat# hor-272) followed, 48h later, by a second injection (5 IU human chorionic gonadotropin - HCG, Peprotech cat#: 100-39-10UG) and then bred to C3H or Pdx1-eGFP or Ins-RFP males. To generate single cells for FACS, embryos at different developmental stages (i.e. e8.25, e10.5, e11.5, e18.5) were dissected out of decidua and collected in 1.5ml tubes containing 1X PBS on ice. For e10.5 and e11.5 embryos, most of the dorsal part (somites) was removed. E18.5 pancreata, clearly visible and easy to distinguish, were dissociated from the rest of the organs. Collected samples were dissociated with 0.05% Trypsin-EDTA (Life Technologies, #25300-054) for 5 min at 37C while gently shaking. The reaction was stopped by adding 0.4 volumes of FBS (Hyclone, SH30071). Explants were homogenized by gentle pipetting. Cells were spun down at 1500rpm for 5min at 4C, and the pellet was resuspended in 320 ul of IMDM (Life Technologies, #12440-053) (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). Twenty (20) ul were transferred into a new tube and 280 ul IMDM were added to it (negative control tube - i.e. samples non incubated with the primary antibody). E8.25 definitive endodermal cells were purified using an antibody detecting ENDM1 (dilution: 1:50) surface antigen (31–33). E10.5 hepatoblasts were sorted from Pdx1-GFP transgenic embryos, staining the cells for the surface marker Liv2 (MBL, cat# D118-3, dilution: 1:100). At the same stage pancreatic precursor Pdx1-GFP positive cells were collected. A subpopulation of Liv2+/Pdx1-GFP+ cells was detected at E10.5, as previously reported (33). These cells were not considered for further analysis. E11.5 hepatoblasts were sorted from wt embryos using the same antibody and conditions as for e10.5 embryos. Cells were incubated for 30-40 min with primary antibody. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 300 ul of IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). PE-conjugated goat anti rat IgG (Biolegend 405406) was added (dilution 1:300). Samples were mixed well and incubated for 30 min on ice in the dark. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 400-800ul 1XPBS + 3% BSA (Sigma A9418-50G). Samples were filtered through a cell strainer (FALCON 5ml polystyrene round-bottom tube with cell-strainer cap, #352235). Samples were kept on ice until sorting. Mature beta cells were isolated from Ins-RFP transgenic embryos as described (34).Two (2) -month old mice were anesthetized using isoflurane (Butler Animal Health Supply, #029405) and monitored by paw pinch.  A V-shaped incision was used to reveal the abdominal cavity, the intestines moved aside, and a 22 or 24 gauge catheter (Midwest Veterinary Supply, #381423) was used to cannulate the venae cavae while liver perfusion media pumps into the liver, and the portal vein is immediately severed. 37°C liver perfusion media (Invitrogen #17701-038) and then liver digest media (Invitrogen #17703-034) are perfused through the liver (45mL each per animal).  Livers are dissociated with a cutting motion with cell scrapers in William’s E Medium (Sigma W4128) and strained through a 100μm filter (BD 352360).   Hepatocytes are loosely pelleted by centrifugation at 50g for 5 min at 4°C. Twenty-five ul of the hepatocytes pellet were transferred into RLT buffer (RNeasy minikit). One (1) month old control and triple knockout (TKO) mutant liver were dissected and cut in pieces. A small piece was transferred to 1.5 ml tube. Samples were minced using dissecting scissors. One (1) ml ACK (Life Technologies, #A10492-01) solution was added and samples were incubated 5-7 min on ice. Samples were spun down at 1,500 rpm for 5 min at 4C; pellet was resuspended in 500 ul 1XPBS and dounced with microcentrifuge tubes pellet pestle (DWK Life Sciences – Kimble - #7495150000) for 10-15 sec on ice. Samples were spun down at 1,500 rpm for 5 min at 4C.Pellet was resuspended in RLT buffer (RNeasy minikit).31.P. Gadue et al., Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm. Stem Cells Dayt. Ohio. 27, 2103–2113 (2009).32.C.-R. Xu et al., Chromatin “prepattern” and histone modifiers in a fate choice for liver and pancreas. Science. 332, 963–966 (2011).33.C.-R. Xu et al., Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification. EMBO J. 33, 2157–2170 (2014).34.D.-S. Li, Y.-H. Yuan, H.-J. Tu, Q.-L. Liang, L.-J. Dai, A protocol for islet isolation from mouse pancreas. Nat. Protoc. 4, 1649–1652 (2009).

tag: developmental stage

value: 4 weeks


title: NC8_iPSC-66017


title: Patsch_EC-66009


title: MSA102_A14_chenodeoxycholic acid 10_uM 6_hours 2011-3-12

tag: perturbagen


title: HUVEC-66001

source_name: umbilical vein

value: endothelial cell (primary cell)

value: unknown


title: MSA101_P22_sotalol 10_uM 6_hours 1275-B-3

value: sotalol

tag: cell id


title: MSA101_P21_sotalol 10_uM 6_hours 3113-6-3

tag: batch


title: MSA102_A05_Chlorpromazine 0.1_uM 6_hours 3121-2-1

value: Chlorpromazine

tag: duration


title: MSA102_A07_arachidonyltrifluoromethane 10_uM 6_hours 3121-2-1

tag: perturbation type


title: HUVEC-66000

value: Ctrl


title: MSA102_A09_diclofenamide 10_uM 6_hours 3121-2-1

value: diclofenamide

tag: well id


title: Definitive Endoderm H3K9me3 ChIP Rep 3

value: Abcam ab8898


title: MSA101_P18_lansoprazole 10_uM 6_hours 1275-B-3

value: lansoprazole

tag: plate id


title: NC8_iPSC-66018

value: iPSC line NC8


title: Definitive Endoderm input for H3K9me3 Rep 1


title: MSA102_A04_dihydroergocristine 1_uM 6_hours 2011-3-12

value: dihydroergocristine

tag: dosage


title: MSA102_A03_dihydroergocristine 1_uM 6_hours 3121-2-1


title: MSA101_P23_Nimodipine 1_uM 6_hours 3113-6-3

value: Nimodipine


title: GSC_MSC_p10_3

city: Haifa

first: Maty

last: Tzukerman

address: Efron, 1

department: Faculty of Medicine

country: Israel

institute: Technion

data_processing:
Quality control was performed using FastQC (v0.11.2) and overrepresented sequences were removed using CUTADAPT tool (v 1.8).
The 100 bp single-end reads were then aligned to Homo sapiens reference genome and annotation file (Homo_sapiens.GRCh38.79 downloaded from ENSEMBL) using TopHat (v2.0.13) allowing 2 mismatches per read with options -very-sensitive and –transcriptome-index
The number of reads per gene was counted using Htseq (0.6.1). Differential expression analysis (DEGs), principal components analysis (PCA) and samples' clustering, were calculated using Deseq2 (version 1.8.1).
DEGs were DEGs were further investigated for function and pathway enrichment using Ingenuity Pathway Analysis (IPA). investigated for function and pathway enrichment using Ingenuity Pathway Analysis (IPA).
Genome_build: GRCh38
Supplementary_files_format_and_content: Excel file includes DESeq2 normalized values for each Sample

source_name: tumor-derived MSC

extract_protocol:
Cells were harvested and RNA was extracted using the RNeasy®Mini Kit (Qiagen, 74104). Agilent TapeStation was used for quality measurements. All samples presented RINe values above 9.5.
12 RNAseq libraries (NEBNext® Ultra™ RNA Library Prep Kit for Illumina, cat no. E7530) were produced according to manufacture protocol using 800 ng total RNA. mRNAs pull-up was performed using Magnetic Isolation Module (NEB cat no. E7490). The libraries were mixed into a single tube with equal molarity of all samples. The RNAseq data was generated using HiSeq2500, two lanes, 100 bp single-end read.

tag: tissue

value: tumor-derived MSC

value: Mesenchymal Stem Cells (MSC)


title: MSA101_P16_levothyroxine sodium 1_uM 6_hours 1275-B-3

value: levothyroxine sodium

value: test


title: MSA102_A06_Chlorpromazine 0.1_uM 6_hours 2011-3-12


title: TKO Liver Rep 1

source_name: TKO 1month old liver


title: MSA101_P17_lansoprazole 10_uM 6_hours 3113-6-3


title: MSA101_P24_Nimodipine 1_uM 6_hours 1275-B-3


title: Ctrl Liver Rep 1

source_name: Ctrl 1month old liver


title: Mature beta cells Rep 2

value: 8 weeks


title: Mature beta cells Rep 1


title: Ctrl Liver Rep 2

value: 4 weeks


title: Immature beta cells Rep 2


title: HUVEC-65999

tag: cell type

value: endothelial cell (primary cell)


title: MSA101_P14_DMSO NA 6_hours 1275-B-3

value: DMSO

value: vehicle


title: GSC_MSC_p7_1


title: MSA101_P15_levothyroxine sodium 1_uM 6_hours 3113-6-3


title: Immature beta cells Rep 1


title: MSA101_P19_sulconazole 8.6_uM 6_hours 3113-6-3

value: sulconazole


title: RP_MSC_M2_3

source_name: reprogrammed MSC

value: reprogrammed MSC


title: MSA101_P20_sulconazole 8.6_uM 6_hours 1275-B-3


title: MSA102_A10_diclofenamide 10_uM 6_hours 2011-3-12

value: diclofenamide


title: MSA101_P12_BUCLADESINE SODIUM 10_uM 6_hours 1275-B-3

value: BUCLADESINE SODIUM


title: MSA101_P13_DMSO NA 6_hours 3113-6-3


title: Pancreatic precursors Rep 2

source_name: Pancreatic precursor (PDX1+ FACS)


title: MSA101_P11_BUCLADESINE SODIUM 10_uM 6_hours 3113-6-3


title: Pancreatic precursors Rep 1


title: RP_MSC_M2_1


title: GSC_MSC_p8_2


title: Hepatocytes Rep 2

source_name: Adult hepatocytes


title: MSA102_A08_arachidonyltrifluoromethane 10_uM 6_hours 2011-3-12

value: arachidonyltrifluoromethane


title: MSA101_P09_niflumic acid 10_uM 6_hours 3113-6-3

value: niflumic acid


title: MSA101_P10_niflumic acid 10_uM 6_hours 1275-B-3


title: RP_MSC_M2_2


title: MSA101_P06_carbimazole 10_uM 6_hours 1275-B-3

value: carbimazole


title: Hepatocytes Rep 1


title: Hepatoblasts Rep 2

source_name: Hepatoblasts (LIV2+ FACS)


title: MSA101_P05_carbimazole 10_uM 6_hours 3113-6-3


title: MSA101_P03_DMSO NA 6_hours 3113-6-3


title: MSA101_P02_salsolidin 1_uM 6_hours 1275-B-3

value: salsolidin


title: T_MSC_3

source_name: naïve MSC

value: naive MSC


title: MSA101_P01_salsolidin 1_uM 6_hours 3113-6-3


title: RP_MSC_M1_2

value: Mesenchymal Stem Cells (MSC)


title: T_MSC_2


title: Hepatoblasts Rep 1


title: RP_MSC_M1_3


title: MSA101_O23_Nimodipine 1_uM 6_hours BJ-2-A

value: Nimodipine


title: RP_MSC_M1_1


title: Definitive Endoderm Rep 2

source_name: Definitiive endoderm (ENDM1+ FACS)


title: MSA101_P04_DMSO NA 6_hours 1275-B-3


title: MSA101_P08_flecainide 8.4_uM 6_hours 1275-B-3

value: flecainide

value: test


title: MSA101_O24_Nimodipine 1_uM 6_hours 2484-2-A


title: Definitive Endoderm Rep 1


title: MSA101_O19_sulconazole 8.6_uM 6_hours BJ-2-A


title: MSA101_O18_lansoprazole 10_uM 6_hours 2484-2-A

value: lansoprazole


title: MSA101_O20_sulconazole 8.6_uM 6_hours 2484-2-A


title: MSA101_O14_DMSO NA 6_hours 2484-2-A


title: MSA101_O22_sotalol 10_uM 6_hours 2484-2-A

value: sotalol


title: T_MSC_1


title: MSA101_O16_levothyroxine sodium 1_uM 6_hours 2484-2-A

value: levothyroxine sodium


title: MSA101_O15_levothyroxine sodium 1_uM 6_hours BJ-2-A


title: MSA101_O17_lansoprazole 10_uM 6_hours BJ-2-A


title: MSA101_O10_niflumic acid 10_uM 6_hours 2484-2-A


title: MSA101_O08_flecainide 8.4_uM 6_hours 2484-2-A


title: MSA101_O13_DMSO NA 6_hours BJ-2-A

value: DMSO

value: vehicle


title: MSA101_O03_DMSO NA 6_hours BJ-2-A


title: MSA101_O04_DMSO NA 6_hours 2484-2-A


title: MSA101_O06_carbimazole 10_uM 6_hours 2484-2-A


title: MSA101_O02_salsolidin 1_uM 6_hours 2484-2-A


title: MSA101_O11_BUCLADESINE SODIUM 10_uM 6_hours BJ-2-A


title: MSA101_O12_BUCLADESINE SODIUM 10_uM 6_hours 2484-2-A


title: MSA101_O01_salsolidin 1_uM 6_hours BJ-2-A


title: MSA101_N20_cephaeline 6_uM 6_hours 1275-B-3

value: cephaeline


title: MSA101_O09_niflumic acid 10_uM 6_hours BJ-2-A


title: MSA101_N21_phenyl biguanide 10_uM 6_hours 3113-6-3

value: phenyl biguanide


title: MSA101_O07_flecainide 8.4_uM 6_hours BJ-2-A


title: MSA101_O05_carbimazole 10_uM 6_hours BJ-2-A


title: MSA101_N22_phenyl biguanide 10_uM 6_hours 1275-B-3


title: MSA101_N19_cephaeline 6_uM 6_hours 3113-6-3


title: MSA101_N24_BAY K8644 1_uM 6_hours 1275-B-3

value: BAY K8644


title: MSA101_N16_trazodone 1_uM 6_hours 1275-B-3

value: trazodone


title: MSA101_N14_pizotifen 1_uM 6_hours 1275-B-3

value: pizotifen


title: MSA101_O21_sotalol 10_uM 6_hours BJ-2-A


title: MSA101_P07_flecainide 8.4_uM 6_hours 3113-6-3


title: MSA101_N18_trichostatin NA 6_hours 1275-B-3

value: trichostatin

value: poscon


title: ATACseq_CAID_3_p14

city: Montréal

first: Gregor

middle: Ulrich

last: Andelfinger

state: QC

address: 3175, Chemin de la Côte-Sainte-Catherine

department: Cardiology

country: Canada

institute: CHU Sainte-Justine

description: ATACseq_raw_readcounts_CAID_syndrome_p8_p14.txt

data_processing:
ATAC-seq reads were trimmed for adapter sequences and low quality score bases and were mapped to the human reference genome. Mapping was performed using BWA-MEM (Li and Durbin, 2009) in paired-end mode at default parameters. Only reads that were properly paired and had an unique alignment (mapping quality > 10) were retained. PCR duplicates were removed using Picard tools.
Peaks were first called on ATAC-seq samples using the MACS2 software suite (Zhang et al., 2008) with the added parameters “g mm -q 0.05 -keep-dup all”. For comparison between samples, all peaks from each sample were merged to provide one set of combined peaks. For quantification of the combined peaks, we used the bamutils count command from NGSUtils (Breese and Liu, 2013) to count the number of overlapping reads for each sample.
Supplementary_files_format_and_content: Tab-delimited text files include raw read counts values for all the samples.

source_name: Human dermal fibroblasts

growth_protocol: Cells were cultured in DMEM, 10% FBS, 1% pen/strep

extract_protocol:
Cells were lysed with ice-cold hypotonic cell lysis buffer (0.1% w/v Sodium Citrate Tribasic Dihydrate and 0.1% v/v Triton X-100) for 30 min on ice followed by a second 30 min lysis step with ice-cold cell lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2, 0.1% v/v IGEPAL CA-630). Transposition reaction was carried out in 25 μl reaction volume using 5 μl of tagmentation DNA enzyme in 2x TD buffer (FC-121-1030, Illumina). EDTA (25mM) was added to the reaction mix before DNA recovery with MiniElute PCR Purification columns (28004, Qiagen).
ATAC-seq libraries were generated from 100,000 cells, as previously described (Buenrostro et al., 2013)

value: Dermal fibroblasts

tag: genotype

tag: passage


title: MSA101_N11_nadolol 10_uM 6_hours 3113-6-3

value: nadolol


title: MSA101_N17_trichostatin NA 6_hours 3113-6-3


title: MSA101_N23_BAY K8644 1_uM 6_hours 3113-6-3


title: MSA101_N13_pizotifen 1_uM 6_hours 3113-6-3


title: ATACseq_CAID_2_p8


title: ATACseq_CAID_2_p14


title: MSA101_N12_nadolol 10_uM 6_hours 1275-B-3


title: ATACseq_control_3_p14

value: wild type


title: ATACseq_CAID_1_p14


title: MSA101_N15_trazodone 1_uM 6_hours 3113-6-3


title: ATACseq_control_3_p8


title: MSA101_M20_cephaeline 6_uM 6_hours 2484-2-A


title: MSA101_N08_DMSO NA 6_hours 1275-B-3


title: MSA101_M21_phenyl biguanide 10_uM 6_hours BJ-2-A


title: MSA101_N02_repaglinide 8.8_uM 6_hours 1275-B-3

value: repaglinide


title: MSA101_N09_liothyronine 3.2_uM 6_hours 3113-6-3

value: liothyronine

value: test


title: MSA101_M23_BAY K8644 1_uM 6_hours BJ-2-A


title: MSA101_N07_DMSO NA 6_hours 3113-6-3


title: MSA101_N06_Quetiapine 10_uM 6_hours 1275-B-3

value: Quetiapine


title: ATACseq_control_2_p14


title: ATACseq_control_1_p14


title: MSA101_M19_cephaeline 6_uM 6_hours BJ-2-A


title: MSA101_M18_trichostatin NA 6_hours 2484-2-A


title: MSA101_N03_niclosamide 10_uM 6_hours 3113-6-3

value: niclosamide


title: MSA101_M24_BAY K8644 1_uM 6_hours 2484-2-A


title: ATACseq_CAID_3_p8


title: MSA101_N05_Quetiapine 10_uM 6_hours 3113-6-3


title: ATACseq_CAID_1_p8


title: MSA101_N10_liothyronine 3.2_uM 6_hours 1275-B-3


title: MSA101_N01_repaglinide 8.8_uM 6_hours 3113-6-3


title: MSA101_M22_phenyl biguanide 10_uM 6_hours 2484-2-A


title: ATACseq_control_1_p8


title: ATACseq_control_2_p8


title: MSA101_M17_trichostatin NA 6_hours BJ-2-A


title: MSA101_M12_nadolol 10_uM 6_hours 2484-2-A


title: MSA101_M16_trazodone 1_uM 6_hours 2484-2-A


title: MSA101_M13_pizotifen 1_uM 6_hours BJ-2-A


title: MSA101_M10_liothyronine 3.2_uM 6_hours 2484-2-A


title: MSA101_M11_nadolol 10_uM 6_hours BJ-2-A


title: MSA101_M09_liothyronine 3.2_uM 6_hours BJ-2-A


title: MSA101_M15_trazodone 1_uM 6_hours BJ-2-A


title: MSA101_M08_DMSO NA 6_hours 2484-2-A

value: DMSO


title: MSA101_M02_repaglinide 8.8_uM 6_hours 2484-2-A


title: MSA101_M05_Quetiapine 10_uM 6_hours BJ-2-A


title: BD patient blood sample in remission  [422820-2]

city: Taipei

first: Ya-Chin

last: Lee

address: Rm 521, No.17, Xu-Zhou Rd.

department: Institute of Epidemiology and Preventive Medicine

country: Taiwan

institute: National Taiwan University

description: Gene expression data were used for intra-individual comparison

hyb_protocol: Following the kit protocol, the cRNA were hybridized on Affymetrix HTA 2.0 array with GeneChip® Hybridization oven 640. Then washed and stained on GeneChip®Fluidics Station 450

scan_protocol: GeneChips were scanned using the GeneChip®Scanner 3000.

data_processing: All of the transcriptome analyses for microarrays were conducted with R software. The microarray data were imported and normalized using Robust Multichip Average (RMA) method in affy package. Then used biomaRt package to match probe ID with Ensembl gene ID using Human Genome Reference GRCh38.

source_name: Whold blood of BD patient in remission

label_protocol: GeneChip™ WT PLUS Reagent Kit were used for the whole process. The intial input was 100 ng RNA.

extract_protocol: Total RNA was extracted from the whold blood with trizol. Used choloroform for lysis, followed by isopropanpl with incubation and centrifugation to get the total RNA precipitates. Then we used 70% ethanol for washing. Finally, we solubilized the RNA pellet in 20-50 μL of RNase-free water and 0.1 mM EDTA for further application

tag: subject status

value: bipolar disorder (BD) patients

value: Male

tag: age

tag: disease status

value: remission

value: Whold blood


title: BD patient blood sample in remission  [423820-2]

tag: gender


title: MSA101_M06_Quetiapine 10_uM 6_hours 2484-2-A


title: BD patient blood sample in manic episode [423120-1]

source_name: Whold blood of BD patient in manic episode

value: Female

value: manic episode


title: MSA101_M14_pizotifen 1_uM 6_hours 2484-2-A

value: pizotifen


title: MSA101_M03_niclosamide 10_uM 6_hours BJ-2-A


title: BD patient blood sample in remission  [423120-2]


title: BD patient blood sample in remission  [422720-2]


title: BD patient blood sample in manic episode [423820-1]


title: BD patient blood sample in manic episode [422820-1]


title: BD patient blood sample in manic episode [422720-1]


title: MSA101_M01_repaglinide 8.8_uM 6_hours BJ-2-A


title: MSA101_M04_niclosamide 10_uM 6_hours 2484-2-A


title: MSA101_L22_bergenin 1_uM 6_hours 1275-B-3

value: bergenin


title: MSA101_L23_MDL100907 0.5_uM 6_hours 3113-6-3


title: MSA101_M07_DMSO NA 6_hours BJ-2-A

value: vehicle


title: BD patient blood sample in remission  [422520-2]


title: MSA101_L21_bergenin 1_uM 6_hours 3113-6-3


title: TCDD-d2-1

city: Research Triangle Park

first: Guang

last: Hu

state: NC

address: 111 TW Alexander DR

department: ESCBL

institute: National Institute of Environmental Health Sciences

data_processing:
RNA-seq reads were first filtered with cutadapt (v1.9.1), and  only those with quality scores higher than 20 were retained for further  analysis. Filtered reads were aligned to reference hg19 with STAR  (v020201) with parameters: –outFilterMismatchNoverLmax 0.04
.
Genome_build: hg19
Supplementary_files_format_and_content: TXT: Genomic features at gene level were generated with featureCounts (v1.5.1)

source_name: embryonic stem cells (ESC)

growth_protocol: Human ESCs were cultured in E8 to 90~100% confluency before induction. To induce cardiac differentiation, the medium was replaced with cardiac differentiation basal medium 1 (RPMI1640 containing 1 × B27 minus insulin supplement) with 5 µM GSK3 inhibitor CHIR99021 (SelleckChem) for 2 days, and changed to basal medium 1 without the inhibitor for another 2 days. Cells were then cultured in basal medium 1 with 5 µM Wnt inhibitor IWR-1 (SelleckChem) for 2 days, followed by basal medium 1 only for 2 additional days. After that, cells were cultured in cardiac differentiation basal medium 2 (RPMI1640 containing 1 × B27 supplement) for 6 more days with medium refreshment every 2 days. During this differentiation process, ESCs were induced to cardiomyocyte through the mesoderm, cardiac mesoderm, cardiomyocyte progenitor, and cardiomyocyte stages

extract_protocol:
Total RNA was isolated using the GeneJet RNA purification kit (Thermo Scientific), and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) to generate cDNA. qPCR was performed using the SsoFast EvaGreen Supermix (Bio-Rad) on the Bio-Rad CFX-384 Real-time PCR Detection System.  Actin (ACTB) was used for normalization.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: 2,3,7,8-Tetrachlorodibenzodioxin (TCDD, Sigma) were dissolved in DMSO. In dose titration, TCDD is added to a final concentration of 2 nM for RNA-seq and ChIP-seq experiments. For control samples, equivalent volume of DMSO is added. The final DMSO concentration in both the control and experimental groups were kept below 0.1% to minimize the impact on cell growth.

tag: cell line

value: Mel1 ES cell line

tag: treatment

value: ESC stage TCDD exposure

tag: time

value: D2 (2-days after the initiation of differentiation by adding inhibitor)


title: TCDD-d2-2


title: TCDD-d1-2

value: D1 (immediately before differentiation)


title: TCDD-d1-1


title: DMSO-d2-2

value: control


title: MSA101_L24_MDL100907 0.5_uM 6_hours 1275-B-3

tag: perturbagen

tag: cell id

tag: batch

value: test


title: MSA101_L20_DMSO NA 6_hours 1275-B-3

tag: duration

tag: perturbation type

tag: well id


title: DMSO-d1-2


title: BD patient blood sample in manic episode [422220-1]

tag: tissue


title: MSA101_L19_DMSO NA 6_hours 3113-6-3

tag: plate id


title: BD patient blood sample in remission  [422220-2]


title: BD patient blood sample in manic episode [422520-1]


title: DMSO-d1-1


title: MSA101_L13_Myeloperoxidase Inhibitor-I 1.1_uM 6_hours 3113-6-3

value: Myeloperoxidase Inhibitor-I

tag: dosage


title: MSA101_L04_tanespimycin NA 6_hours 1275-B-3

value: tanespimycin

value: poscon


title: MSA101_L12_arcaine 10_uM 6_hours 1275-B-3

value: arcaine


title: MSA101_L11_arcaine 10_uM 6_hours 3113-6-3


title: MSA101_L18_celastrol 2.5_uM 6_hours 1275-B-3

value: celastrol


title: MSA101_L16_PK11195 1_uM 6_hours 1275-B-3


title: MSA101_L14_Myeloperoxidase Inhibitor-I 1.1_uM 6_hours 1275-B-3


title: MSA101_L06_ronidazole 1_uM 6_hours 1275-B-3

value: ronidazole


title: MSA101_L09_Amoxapine 0.1_uM 6_hours 3113-6-3

value: Amoxapine


title: MSA101_L05_ronidazole 1_uM 6_hours 3113-6-3


title: MSA101_L17_celastrol 2.5_uM 6_hours 3113-6-3


title: MSA101_L15_PK11195 1_uM 6_hours 3113-6-3


title: AhR-ChIP-rep2

data_processing:
Filtered reads were aligned with bowtie(v1.1.2) with parameters: -v 2 -m 1 –best –strata
bigWig: bedtools genomeCoverageBed was used for generate bedGraph file, coverage then normalized to reads per 10 million aligned reads, bedGraphToBigWig was used to produce final bigWig
Genome_build: hg19
Supplementary_files_format_and_content: bigWig

extract_protocol:
Human ESC cells were fixed using 1% formaldehyde for 10 min, and followed by 0.125 M glycine 5 min to stop the fixation. Then the cells were harvested, and DNA was fragmented to 300–500 bp by sonication with a microtip attached to Misonix 3000 sonicator. Immunoprecipitation was performed with 3 mg Dynabeads protein G (Life Technology) and AhR antibody overnight at 4C. Afterward, beads were washed, eluted, and reverse cross-linked. DNA was extracted by phenol/chloroform and precipitated.
For ChIP-seq, 1 ng precipitated DNA or input was used to generate DNA library by use of Nextera XT DNA sample preparation Kit (Illumina) according to the manufacturer’s instruction.

value: TCDD

tag: chip antibody

value: Ah Receptor Antibody (H-211) (Santa Cruz, catalog# sc-5579)


title: AhR-Input-rep1


title: MSA101_L10_Amoxapine 0.1_uM 6_hours 1275-B-3


title: MSA101_L07_isoniazid 10_uM 6_hours 3113-6-3

value: isoniazid


title: AhR-ChIP-rep1


title: MSA101_L03_tanespimycin NA 6_hours 3113-6-3


title: AhR-Input-rep2


title: MSA101_K19_DMSO NA 6_hours BJ-2-A

value: DMSO

value: vehicle


title: MSA101_K24_MDL100907 0.5_uM 6_hours 2484-2-A


title: MSA101_K20_DMSO NA 6_hours 2484-2-A


title: DMSO-d2-1

value: Mel1 ES cell line


title: MSA101_L02_budesonide 9.2_uM 6_hours 1275-B-3

value: budesonide


title: MSA101_K23_MDL100907 0.5_uM 6_hours BJ-2-A


title: MSA101_L01_budesonide 9.2_uM 6_hours 3113-6-3


title: MSA101_K22_bergenin 1_uM 6_hours 2484-2-A

value: bergenin


title: K562 cell ATAC-seq replicate 2

city: Ithaca

first: Charles

middle: G

last: Danko

state: NY

address: Hungerford Hill Rd

department: Baker Institute

institute: Cornell University

description:
ATAC-seq
Tn5 accessible DNA

data_processing:
Data was aligned to the reference genome using scripts here: https://github.com/Danko-Lab/utils/tree/master/proseq
Genome_build: hg19
Supplementary_files_format_and_content: Mapped reads are provided in bigWig format.

source_name: K562 cells

extract_protocol:
K562 cells were grown in RPMI-1640 media. Nuclei were extracted as described (Danko et. al. (2015) Nature Methods).
We prepared PRO-seq libraries as described in Kwak et. al. (2013) Science. ATAC-seq libraries were prepared as described Buenrostro et. al. (2013) Nature Methods.


title: K562 cell ATAC-seq replicate 1

description:
ATAC-seq
Tn5 accessible DNA


title: MSA101_K18_celastrol 2.5_uM 6_hours 2484-2-A


title: K562 cell PRO-seq

description:
PRO-seq
nascent RNA

data_processing:
Library strategy: PRO-seq
Data was aligned to the reference genome using scripts here: https://github.com/Danko-Lab/utils/tree/master/proseq
Genome_build: hg19
Supplementary_files_format_and_content: Mapped reads are provided in bigWig format.


title: Bulk Hi-C Library WTC-11 CM Rep 2

city: Seattle

last: Murry

state: WA

address: 850 Republican St

department: Institute for Stem Cell and Regenerative Medicine

institute: University of Washington

data_processing:
Reads were mapped with BWA-MEM version 0.7.13 to hg38 using default parameters. Secondary mappings were filtered out with samtools.
Primary mappings were processed using HIC-PRO version 2.7.6, excluding pairs less than 1KB apart and excluding MAPQ scores <30, to generate validPairs.
validPair files were used as input for HOMER version 4.7 at 500KB resolution to generate PC1 values (A/B compartment calls).
Genome_build: hg38
Supplementary_files_format_and_content: validPairs file is processed output from HiC-Pro and contains read name and mapping location for the two reads after passing distance and quality threshold
Supplementary_files_format_and_content: PC1.bedGraph files are the ouput from HOMER on the processed Hi-C data, representing the A/B compartment calls, in bedGraph format (coordinates and score)

source_name: Cultured Cell Lines

growth_protocol: WTC-11 hiPSCs were maintained on lamin-521 matrix (Biolamina) at 0.5mg/cm2 with Essential 8 (E8) media (ThermoFisher).For differentiation, 4x106 cells in E8 with 10mM Y-27632 (Tocris) were seeded per well of a 12-well dish that was pre-coated with 2mg/cm2 lamin-521. After 24 hours media was changed to E8 with 1mM CHIR-99021 (Cayman), denoted day -1. On day 0 media was changed to RPMI (ThermoFisher) with BSA (500mg/ml) (Sigma, A9418) + ascorbic acid (213mg/ml) (Sigma, A8960), denoted RBA media, supplemented with 3mM CHIR-99021. Samples for day 0 were collected prior to media change. Day 2 samples were collected 48 hours later. Wells for further differentiation were left unchanged until day 3, when media was changed to RBA plus 1mM Xav-939 (Tocris). Day 5, samples were either collected or changed to RBA without inhibitors. On day 7 media was changed to RPMI plus B27 (Thermo Fisher, 17504044), with further media changes every other day until day 14 samples were collected. Fetal Heart samples were collected by the Laboratory of Developmental Biology at the University of Washington (UW), in accordance with IRB approval from the UW. Collected hearts were washed in PBS and flash frozen in liquid N2 and stored at -80 prior to further analysis.

extract_protocol:
Cells are lysed using 10mM Tris-HCl, pH 8.0, 10mM NaCl, 0.2% Igepal CA-630, 1x Protease Inhibitor in ddH2O
Library prep was carried out using a similar protocol to the insitu DNase Hi-C protocols (V. Ramani et al. Nat Protoc. 2016). Samples were barcoded during final library PCR and samples were demultiplexed prior to downstream analysis.

treatment_protocol: Cells were fixed using 2% formaldehyde for 10 minutes. For fetal hearts samples were first treated with a dismembrator for 1 min at 2000 rpm and then fixed in 1% formaldehyde for 25 minutes.

tag: cell type

value: cardiomyocyte (CM)

tag: time point

value: Day 14 of differentiation


title: Bulk Hi-C Library WTC-11 CM Rep 1


title: Bulk Hi-C Library WTC-11 CP Rep 2

value: cardiac progenitor (CP)

value: Day 5 of differentiation


title: MSA101_K21_bergenin 1_uM 6_hours BJ-2-A

value: test


title: MSA101_K13_Myeloperoxidase Inhibitor-I 1.1_uM 6_hours BJ-2-A

value: Myeloperoxidase Inhibitor-I


title: Bulk Hi-C Library WTC-11 CP Rep 1


title: MSA101_K10_Amoxapine 0.1_uM 6_hours 2484-2-A


title: MSA101_K17_celastrol 2.5_uM 6_hours BJ-2-A

value: celastrol


title: MSA101_K11_arcaine 10_uM 6_hours BJ-2-A

value: arcaine


title: MSA101_K15_PK11195 1_uM 6_hours BJ-2-A


title: MSA101_K16_PK11195 1_uM 6_hours 2484-2-A


title: MSA101_K12_arcaine 10_uM 6_hours 2484-2-A


title: MSA101_K14_Myeloperoxidase Inhibitor-I 1.1_uM 6_hours 2484-2-A


title: MSA101_K09_Amoxapine 0.1_uM 6_hours BJ-2-A

value: Amoxapine


title: MSA101_K07_isoniazid 10_uM 6_hours BJ-2-A


title: MSA101_K06_ronidazole 1_uM 6_hours 2484-2-A

value: ronidazole


title: Bulk Hi-C Library WTC-11 MES Rep 2

value: mesoderm (MES)

value: Day 2 of differentiation


title: Bulk Hi-C Library WTC-11 MES Rep 1


title: HEK_and_S2_RNA_mixture_in_100:1

city: Solna

first: Wenjing

last: Kang

address: SciLifeLab, Tomtebodavägen 23

country: Sweden

institute: SciLifeLab

data_processing:
Basecalls performed using bcl2fastq2 conversion software v 2.17 with parameter “--barcode-mismatches 0”
All FASTQ files were processed using QC mode of miRTrace
Supplementary_files_format_and_content: tab-delimited text files include PHRED scores, read lengths, QC status, RNA types, miRNA complexities, basic and detailed contamination information for each sample.

organism:
Drosophila melanogaster
Homo sapiens

extract_protocol:
Trizol
Libraries were prepared based on Illumina TruSeq small RNA kit v2. 1 μg of HEK-293T and Drosophila S2 total RNA was used for standard small RNA library preparation. Contaminated HEK-293T small RNA libraries were generated by pre-mixing HEK-293T total RNA with the total RNA isolated from S2 cells. The following ratios of HEK-293T to S2 total RNA were used 10:1, 100:1, 1000:1. 50 ng of HEK-293T total RNA were used for preparation of low input small RNA library. 1 μg of RNase A treated total RNA for 5 min at room temperature was used for preparation of degraded small RNA library. Single-end sequencing was carried out on an Illumina NextSeq 500.

value: HEK-293T and S2

value: cross-species contamiantion


title: HEK_and_S2_RNA_mixture_in_10:1


title: HEK_and_S2_RNA_mixture_in_1000:1


title: Bulk Hi-C Library WTC-11 PSC Rep 2

value: pluripotent stem cell (PSC)

value: Day 0 of differentiation


title: MSA101_K01_budesonide 9.2_uM 6_hours BJ-2-A

value: budesonide


title: MSA101_K08_isoniazid 10_uM 6_hours 2484-2-A

value: isoniazid


title: Bulk Hi-C Library WTC-11 PSC Rep 1


title: MSA101_K02_budesonide 9.2_uM 6_hours 2484-2-A


title: MSA101_K04_tanespimycin NA 6_hours 2484-2-A

value: tanespimycin

value: poscon


title: MSA101_K03_tanespimycin NA 6_hours BJ-2-A


title: Degraded_HEK_RNA

value: RNase A digestion


title: Low-input_HEK_RNA

value: sample dilution


title: MSA101_J23_TCB-2 0.1_uM 6_hours 3113-6-3

value: test


title: MSA101_K05_ronidazole 1_uM 6_hours BJ-2-A


title: MSA101_J22_lycorine 10_uM 6_hours 1275-B-3

value: lycorine


title: HEK_control

value: control


title: MSA101_J18_Ziprasidone 0.1_uM 6_hours 1275-B-3

value: Ziprasidone


title: MSA101_J19_diltiazem 3_uM 6_hours 3113-6-3

value: diltiazem


title: HBOC-234

type: genomic

city: Hannover

first: Doris

last: Steinemann

address: Carl-Neuberg-Straße 1

department: Human Genetics

country: Germany

institute: Medizinische Hochschule Hannover

hyb_protocol: According to manufracturers recommodations: Agilent Oligonucleotide Array-Based CGH for Genomic DNA analysis. Enzymatic labeling for blood, cells, or tissues (with a high throughput option) protocol Version (7.5, June 2016)

data_processing:
The raw data was processed by feature extraction version 11.0.1.1 (Agilent Technologies)
Aberrations were detected with the ADM-2 algorithm and thresholds of 6 and 2log02 (Cy3/Cy5) in Genomic Workbench (version 7.0.4.0, Agilent Technologies)

extract_protocol: Genomic DNA was extracted from EDTA blood samples by QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany)

tag: subject status

value: BRCA1/2 negative Hereditary breast and/or ovarian cancer (HBOC) patient

value: blood

source_name: Megapool Reference DNA (female) EA-100F (Kreatech Biotechnology, Amsterdam, The Netherlands)

tag: sample type

value: reference DNA


title: MSA101_J16_DMSO NA 6_hours 1275-B-3

value: DMSO

value: vehicle


title: MSA101_J24_TCB-2 0.1_uM 6_hours 1275-B-3


title: MSA101_J17_Ziprasidone 0.1_uM 6_hours 3113-6-3


title: HBOC-237

scan_protocol: According to manufracturers recommodations: Agilent Oligonucleotide Array-Based CGH for Genomic DNA analysis. Enzymatic labeling for blood, cells, or tissues (with a high throughput option) protocol Version (7.5, June 2016)


title: MSA101_J13_suloctidil 10_uM 6_hours 3113-6-3

value: suloctidil


title: HBOC-235

label_protocol: According to manufracturers recommodations: Agilent Oligonucleotide Array-Based CGH for Genomic DNA analysis. Enzymatic labeling for blood, cells, or tissues (with a high throughput option) protocol Version (7.5, June 2016)


title: HBOC-236


title: MSA101_J20_diltiazem 3_uM 6_hours 1275-B-3


title: HBOC-233


title: MSA101_J11_vigabatrin 10_uM 6_hours 3113-6-3

value: vigabatrin


title: MSA101_J21_lycorine 10_uM 6_hours 3113-6-3


title: MSA101_J10_spiradoline 0.1_uM 6_hours 1275-B-3

value: spiradoline


title: MSA101_J12_vigabatrin 10_uM 6_hours 1275-B-3


title: MSA101_J08_chlorcyclizine 1_uM 6_hours 1275-B-3

value: chlorcyclizine


title: MSA101_J15_DMSO NA 6_hours 3113-6-3


title: MSA101_J09_spiradoline 0.1_uM 6_hours 3113-6-3


title: MSA101_J14_suloctidil 10_uM 6_hours 1275-B-3


title: A2

city: Chiayi

first: Shou-Tsung

last: Wang

address: No. 300, Syuefu Rd.

institute: National Chiayi University

hyb_protocol: Fluorescent targets were hybridized to the Mouse Whole Genome OneArray® with Phalanx hybridization buffer using Phalanx Hybridization System. After 16 hrs hybridization at 50 ℃, non-speciﬁc binding targets were washed away by three different washing steps (Wash I 42 °C 5 mins；Wash II 42 °C, 5 mins, 25 °C 5 mins; Wash III rinse 20 times)

scan_protocol: The arrays were scanned by Agilent G2505C scanner and quantify the fluoresence intensity.

data_processing:
The signal intensity of each spot was loaded into Rosetta Resolver System® (Rosetta Biosoftware) to process data analysis. The error model of Rosetta Resolver System® could remove both systematic and random errors from the data. We ﬁltered out spots that the ﬂag is less than 0. Spots that passed the criteria were normalized by 50% media scaling normalization method. Normalize intensities: Median scaling performed on data set without flagged and control data. Merge technical replicate data: Average intensity values calculated on technical replicates.
Calculate average ratios of the hybridization replicates and find significant values for each gene by pairwise comparision (Ranking and hypothesis testing).

source_name: urinary bladders

label_protocol: 1 µg of total RNA was reverse-transcribed and amplified using Amino Allyl aRNA Amplification Kit (Phalanx Biotech Group, Taiwan) and Cy5 dyes (Amersham Pharmacia, Piscataway, NJ, USA).

extract_protocol: RNA extraction by Trizol. RNA purity was checked by optical density of NanoDrop ND-1000 and agarose electrophoresis with the result of A260/A280≧1.8, no gDNA contamination. RNA integrity was measured by Agilent RNA 6000 Nano Assay (RIN≧8).

value: urinary bladders

tag: strain

tag: gender

value: female

value: 50 ppm arsenic-contained RO water


title: A1

tag: treatment


title: MSA101_J07_chlorcyclizine 1_uM 6_hours 3113-6-3

tag: perturbagen

value: test


title: C2

value: RO water


title: HBOC-232


title: HBOC-231


title: S2_control

organism: Drosophila melanogaster

tag: cell line

value: control


title: C1


title: HBOC-230


title: HBOC-228


title: HBOC-229


title: Urea 19

city: Karise

first: Rune

middle: Nørgaard

last: Rasmussen

address: Vesterled 4

department: Drug transporters in ADME

country: Denmark

institute: Southern University of Denmark

description:
Single read RNA-sequencing
Poly A RNA
processed data file: Count.table.original.featurecounts.txt

data_processing:
Basecallling by CASAVA.
Alignment was performed with Hisat 2.0.5.
Read count was performed using featureCounts v1.5.1.
Genome_build: CFA 3.1
Supplementary_files_format_and_content: Count.table.original.featurecounts.txt: Tab-delimited text file includes counts.

organism: Canis lupus familiaris

source_name: Madin-Darby Canine Kidney (MDCK) I cells

growth_protocol: MDCK I cells were cultured using DMEM/F-12 containing 1% penicillin-streptomycin, 2 mM L-glutamine and 10% FBS in an atmosphere of 5% CO2- 95% O2 at 37 °C at a density of 7.6 x 10.000 in non-coated 24 well plates for a total of 144 h. The cells were treated with different compounds in the last 24 h of the cultivation.

extract_protocol:
RNA was isolated using Nucleospin® RNA/Protein following the protocol of the manufacturer.
The cDNA library was constructed with TruSeq RNA Library Preparation Kit v2 from Illumina following the protocol of the manufacturer.

treatment_protocol: MDCK I cells were treated for 24 h with medium supplemented with 200 mOsm raffinose, NaCl or urea (total of 500 mOsm). The control was regular medium (300 mOsm).

value: MDCK I

tag: cell passage

value: 200 mOsm Urea (500 mOsm)


title: Urea 17


title: Urea 14


title: HBOC-227

value: BRCA1/2 negative Hereditary breast and/or ovarian cancer (HBOC) patient


title: MSA101_J06_trimethobenzamide 1_uM 6_hours 1275-B-3

data_processing: Raw intensity measurements are deconvoluted to provide intensity values for each landmark gene (the 978 directly measured features). Data is then normalized using a calibration curve based on invariant gene sets, and then quantile normalized.

value: trimethobenzamide

tag: cell id

tag: batch

tag: duration


title: Urea 16


title: HBOC-225

tag: tissue

value: blood

value: reference DNA


title: MSA101_J05_trimethobenzamide 1_uM 6_hours 3113-6-3

tag: perturbation type

tag: well id

tag: plate id


title: HBOC-226


title: MSA101_J04_mebendazole 10_uM 6_hours 1275-B-3

value: mebendazole

tag: dosage


title: Urea 13


title: HBOC-224


title: HBOC-222


title: Urea 11


title: MSA101_J03_mebendazole 10_uM 6_hours 3113-6-3


title: HBOC-223


title: Urea 7


title: MSA101_J01_DMSO NA 6_hours 3113-6-3

value: DMSO

value: vehicle


title: MSA101_J02_DMSO NA 6_hours 1275-B-3


title: HBOC-220


title: MSA101_I24_TCB-2 0.1_uM 6_hours 2484-2-A


title: Urea 8


title: MSA101_I23_TCB-2 0.1_uM 6_hours BJ-2-A


title: HBOC-218


title: Urea 1


title: Raffinose 19

value: 200 mOsm raffinose (500 mOsm)


title: MSA101_I20_diltiazem 3_uM 6_hours 2484-2-A

value: diltiazem


title: Urea 10


title: Urea 5


title: HBOC-221


title: MSA101_I21_lycorine 10_uM 6_hours BJ-2-A

value: lycorine


title: MSA101_I17_Ziprasidone 0.1_uM 6_hours BJ-2-A

value: Ziprasidone


title: HBOC-215


title: MSA101_I18_Ziprasidone 0.1_uM 6_hours 2484-2-A


title: HBOC-212


title: Urea 4


title: Raffinose 16


title: HBOC-217


title: HBOC-219


title: Raffinose 13


title: HBOC-213


title: MSA101_I19_diltiazem 3_uM 6_hours BJ-2-A


title: MSA101_I22_lycorine 10_uM 6_hours 2484-2-A


title: MSA101_I16_DMSO NA 6_hours 2484-2-A


title: Raffinose 11


title: MSA101_I14_suloctidil 10_uM 6_hours 2484-2-A

value: suloctidil


title: Raffinose 8


title: Raffinose 17


title: Raffinose 10


title: HBOC-214


title: MSA101_I11_vigabatrin 10_uM 6_hours BJ-2-A

value: vigabatrin

value: test


title: MSA101_I13_suloctidil 10_uM 6_hours BJ-2-A


title: Raffinose 14


title: MSA101_I15_DMSO NA 6_hours BJ-2-A


title: MSA101_I12_vigabatrin 10_uM 6_hours 2484-2-A


title: MSA101_I10_spiradoline 0.1_uM 6_hours 2484-2-A

value: spiradoline


title: HBOC-210


title: HBOC-209


title: HBOC-216


title: Raffinose 7

value: MDCK I


title: HBOC-207


title: Raffinose 5


title: Raffinose 1


title: HBOC-206


title: Raffinose 4


title: Natrium 17

value: 200 mOsm NaCl (500 mOsm)


title: HBOC-208

value: BRCA1/2 negative Hereditary breast and/or ovarian cancer (HBOC) patient


title: MSA101_I06_trimethobenzamide 1_uM 6_hours 2484-2-A

value: trimethobenzamide


title: MSA101_I09_spiradoline 0.1_uM 6_hours BJ-2-A


title: Natrium 16


title: MSA101_I07_chlorcyclizine 1_uM 6_hours BJ-2-A

value: chlorcyclizine


title: MSA101_I05_trimethobenzamide 1_uM 6_hours BJ-2-A


title: MSA101_I08_chlorcyclizine 1_uM 6_hours 2484-2-A


title: HBOC-205

value: blood

value: reference DNA


title: MSA101_I04_mebendazole 10_uM 6_hours 2484-2-A

value: mebendazole


title: HBOC-202


title: Natrium 13


title: MSA101_I03_mebendazole 10_uM 6_hours BJ-2-A


title: HBOC-204


title: HBOC-199


title: HBOC-200


title: Natrium 14


title: Natrium 19


title: HBOC-201


title: MSA101_I02_DMSO NA 6_hours 2484-2-A

value: DMSO

value: vehicle


title: HBOC-203


title: Natrium 10


title: Natrium 8


title: Natrium 11


title: HBOC-198


title: Natrium 7


title: HBOC-196


title: Natrium 5


title: MSA101_I01_DMSO NA 6_hours BJ-2-A


title: MSA101_H24_DMSO NA 6_hours 1275-B-3


title: MSA101_H23_DMSO NA 6_hours 3113-6-3


title: Natrium 4


title: Natrium 1


title: Control 19

value: Control (300 mOsm)


title: Control 16


title: MSA101_H21_3-acetamidocoumarin 10_uM 6_hours 3113-6-3


title: Control 13


title: MSA101_H22_3-acetamidocoumarin 10_uM 6_hours 1275-B-3


title: Control 11


title: Control 17


title: MSA101_H20_DL-thiorphan 1_uM 6_hours 1275-B-3

value: DL-thiorphan


title: MSA101_H18_Prestwick-675 10_uM 6_hours 1275-B-3


title: Control 14


title: Control 10


title: MSA101_H19_DL-thiorphan 1_uM 6_hours 3113-6-3


title: MSA101_H17_Prestwick-675 10_uM 6_hours 3113-6-3


title: HBOC-191


title: HBOC-195


title: MSA101_H16_prenylamine 9.6_uM 6_hours 1275-B-3

value: prenylamine


title: HBOC-194