title: M397_Day3_K27_H3K4me3_ChIPSeq_treated_with_v3

status: Public on May 12 2020

city: Los Angeles

first: Xiang

last: Lu

address: 10833 Le Conte Ave

department: Department of Molecular and Medical Pharmaacology

institute: University of California, Los Angeles

data_processing:
Reads were aligned to hg19 with bowtie2
Peaks were called on the merged set of bam files using MACS2
Differential accessibility or enrichment of histone mark were measured using diffReps/DESeq2
Differentially accessible or enrichmental regions were called if the absolute log value of the fold change was more than 1 at an FDR <0.05
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files. To visualize peaks in each sample, bed graph file was generated using MACS2 with following parametes: --nomodel, --broad, --bdg, --SPMR. Then the generated bed graph file was converted into bigwig file by bedGraphToBigWig tool

growth_protocol: M-series patient derived cell lines used in this study were generated under UCLA institutional review board approval # 11–003254. Cells were cultured in a water-saturated incubator at 37 °C with 5% CO2 in RPMI 1640 with L-glutamine (Life Technologies), supplemented with 10% fetal bovine serum (Omega), and 0.2% antibiotics (MycoZapTM Plus-CL from Lonza). The cell lines were tested for mycoplasma and were periodically authenticated to its early passage using GenePrint 10 System (Promega).

extract_protocol:
Briefly, cells were cultured to ~80% confluency in a petri dish containing 10 mL of growth media, and then fixed in 1% formaldehyde by adding 275 μl of 37% formaldehyde for 10 minutes to cross-link protein–DNA complexes at room temperature. The unreacted formaldehyde was quenched by adding Glycine to a final concentration 0.125 M. Gently swirl dish to mix. The nuclear pellet was isolated with Cell Lysis Buffer. The pellet was resuspended with 500 μl SDS Lysis Buffer containing 1X Protease Inhibitor Cocktail II before sonication for 4 min (10 s on, 30 s off, 10% strength in a Bioruptor to yield DNA fragments of 0.2-1.0 kb in length. The lysates were cleared by centrifugation (12,000g for 10 min at 4 °C) and diluted tenfold in ChIP dilution buffer to decrease the concentration of SDS. After keeping 10% of the sample as input, 500 μl supernatant was incubated overnight at 4 °C with antibody and 20 μL of fully resuspended protein A/G magnetic beads. The washing, elution, reverse cross-linking, and purification steps were performed according to manufacturer’s description. ChIPed Eluted DNA was quantified by Qubit dsDNA HS Assay Kit.
ChIP-seq libraries were prepared with Kapa DNA HyperPrep Kit (Kapa, Cat KK 8700) according to the manufacturer's protocol. Briefly, 5-10 ng of immunoprecipitated DNA was underwent end-repaired, A tailing and adaptor ligation. A 10 cycles of PCR was performed to produce the final sequencing library. The libraries were validated with the Agilent Bioanalyzer DNA High Sensitivity Kit, and quantified with qPCR.

treatment_protocol: BRAF inhibitor (vemurafenib) from Selleck Chemicals LLC, were dissolved in DMSO at designated concentrations before applying to cell culture media. Cells were plated in 10cm tissue culture plate at 60% confluency and treated with certain drugs for the specified numbers of days. M397 cells were treated with 3uM of vemurafenib for 59days and at day29, drug were removed and cells were cultured with normal medium for another 35days.

tag: cell type

value: Patient-derived melanoma cell lines

tag: cell line

tag: chip antibody

value: H3K4me3 (Millipore, 07-473)


title: M397_Drug_removal_K27_H3K27ac_ChIPSeq

type: SRA

state: CA

country: USA

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

value: H3K27ac (Millipore, 17-683)


title: M397_Day32_K27_H3K27ac_ChIPSeq


title: M397_Day3_K4_H3K4me3_ChIPSeq_treated_with_v3


title: M397_Day3_K4_H3K4me3_ChIPSeq


title: M397_Day32_K4_H3K4me3_ChIPSeq


title: M397_Day0_K27_H3K27ac_ChIPSeq


title: M397_Drug_removal K4_H3K4me3_ChIPSeq


title: Sample 6: POL1 High/LGR5 negative

status: Public on May 22 2020

type: RNA

city: Sabadell

first: Antoni

last: Berenguer-LLergo

state: Barcelona

address: Parc del Taulí, 1

department: A8G2 Group - Rheumatology Service

country: Spain

institute: Parc Taulí Research and Innovation Institute (I3PT)

description: POL1 High/LGR5 negative cells from CRC organoids sorted by FACS

hyb_protocol: cDNA was hybridized to the microarray for 16 hours at 45ºC, washing and staining of microarrays was performed using a GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA)

scan_protocol: Arrays were scanned with GeneChip scanner GSC3000 (Affymetrix, Santa Clara, CA)

data_processing: RMA normalization method as implemented in affy Bioconductor R package

label: biotin

molecule: total RNA

source_name: FACS-sorted cells from CRC organoids

label_protocol: 8 µg of cDNA was subsequently fragmented and labeled using GeneChip Mapping 250K Nsp Assay Kit (Affymetrix; catalog # 900766), according to manufacturer's instructions.

growth_protocol: Genome edited organoids were injected subcutaneously into immunodecifient animals. Tumor growth was monitorized and tumors were ressected when they reached and average size of 100mm3

extract_protocol: RNA was isolated using magnetic beads from 500 and 1000 cels. Library preparation and amplification were performed as described by Gonzalez et al., 2010. Amplification was performed for 21 and 24 cycles respectively.

treatment_protocol: No treatment

tag: sample id

value: POL1 High

value: LGR5 negative


title: Sample 8: POL1 Low/LGR5 negative

description: POL1 Low/LGR5 negative cells from CRC organoids sorted by FACS

value: POL1 Low


title: M397_Day3_K27_H3K27ac_ChIPSeq


title: VE-cadherin -/- plus control

status: Public on May 07 2020

city: Camperdown

first: Jennifer

middle: R

last: Gamble

state: New South Wales

address: Building 93, Royal Prince Alfred Hospital

department: Vascular Biology

country: Australia

institute: CENTENARY INSTITUTE

description:
transformed cell line
VE-cadherin null EC
Jial01
R1 fastq unavailable.  Only the R2 fastq is available.

library_source: transcriptomic

data_processing:
Library preparations were sequenced on an Illumina Hiseq4000 platform and 150 bp paired-end reads were generated
Reference genome and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly. Indices of the reference genome were built using Bowtie (v2.2.3) and paired-end clean reads were aligned to the reference genome using TopHat (v2.0.12).
HTSeq (v0.6.1) was used to count the read numbers mapped of each gene, followed by calculation of RPKM (Reads Per Kilobase of exon model per Million mapped reads) of each gene based on the length of the gene and reads count mapped to that gene
Differentially expressed genes were then determined using DESeq R package (v1.12.0). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted P-value<0.05 and FDR<0.005 were assigned as differentially expressed.
Genome_build: hg19; mm10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

organism: Mus musculus

source_name: VE-cadherin null EC

extract_protocol:
Total RNA was extracted from ECs using RNeasy Plus Mini Kit (Qiagen)
RNA-Seq libraries for mRNA profiling were prepared from 1.5-5  µg total RNA using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations

treatment_protocol: ECs were treated with either control or CD5-2 for 24h prior to RNA extraction

value: VE-cadherin null EC

tag: genotype

value: VE-cadherin -/-

tag: treatment

value: control


title: Sample 5: POL1 High/LGR5 Positive

description: POL1 High/LGR5 Positive cells from CRC organoids sorted by FACS

value: LGR5 Positive


title: Sample 3: POL1 Low/LGR5 Positive

description: POL1 Low/LGR5 Positive cells from CRC organoids sorted by FACS


title: Sample 2: POL1 High/LGR5 negative


title: M397_Day0_K4_H3K4me3_ChIPSeq


title: Sample 7: POL1 Low/LGR5 Positive


title: OmniATAC-seq_Dog_Dog-OralMel-18249_BL

status: Public on Apr 22 2020

city: Leuven

first: Stein

last: Aerts

address: O&N4 Herestraat 49 PO Box 602

country: Belgium

institute: KU Leuven

data_processing:
Data processing of human melanoma baseline OmniATAC-seq samples: Paired-end reads were mapped to the human genome (hg19-Gencode v18) using bowtie2 (v2.2.6). Mapped reads were sorted using SAMtools (v1.8) and duplicates were removed using Picard MarkDuplicates (v1.134). Reads were filtered by removing chromosomal reads and filtering for Q>30 using SAMtools. Bam files of technical replicates of the same cell line were merged at this point using samtools merge. Peaks were called using MACS2 (v2.1.2) callpeak using the parameters -q 0.05, --nomodel, --call-summits, --shift -75 --keep-dup all and --extsize 150 per sample. Chromosomal peaks, blacklisted regions (ENCODE) and peaks overlapping with alternative chromosomes were removed. Summits were extended by 250bp up- and downstream using slopBed (bedtools; v2.28.0), providing human chromosome sizes. Peaks were normalised for the library size using a custom script and overlapping peaks were filtered using the peak score by keeping the peak with the highest score. For visualisation in IGV, normalised bigWigs were made by bamCoverage (Deeptools, v3.3.1), using as parameters --normalizeUsing None, -bl EncodeBlackListedRegions --effectiveGenomeSize 2913022398 and as scaling parameter (-scaleFactor) 1/(RIP/1E6), where the RIP stands for the number of reads in peaks.
Data processing of non-human (Omni)ATAC-seq samples, and of human SOX10 and TFAP2A knock-down OmniATAC-seq data: Adapter sequences were trimmed from the fastq files using fastq-mcf (as part of eautils; v1.05) and the read quality was checked using FastQC (v0.11.8). Reads were mapped using STAR (v2.5.1b) (for the zebrafish samples paired-end reads were mapped) to the genome which were downloaded from UCSC (http://hgdownload.cse.ucsc.edu/goldenPath/) (for human: hg19-Gencode v18; for dog: canFam3; for horse: equCab2; for pig: susScr11; for mouse: mm10; for zebrafish: danRer10) and by applying the parameters --alignIntronMax 1 and --aslignIntronMin 2. Mapped reads were filtered for quality using SAMtools (v1.2) view with parameter –q4, sorted with SAMtools sort and indexed using SAMtools index. Peaks were called using MACS2 (v2.1.2) callpeak using the parameters -q 0.05, --nomodel, --call-summits, --shift -75 --keep-dup all and with the genome size for the correct species in --g, and this for each sample per species separately. Summits were extended by 250bp up- and downstream using slopBed (bedtools; v2.28.0), providing the chromosome sizes for the specific species. Per sample, peaks were normalised for the library size using a custom script and overlapping peaks were filtered using the peak score (keeping the highest scoring peak). Normalised bedGraphs were produced by genomeCoverageBed (as part of bedtools; v2.28.0) using as scaling parameter (-scale) 1E6/(number of non-chromosomal mapping reads). BedGraphs were converted to bigWigs by the bedtools suit functions bedSort to sort the bedGraphs, followed by bedGraphToBigWig to create the bigWigs, which were used in IGV for visualisation.
Genome_build: For human: hg19-Gencode v18; for dog: canFam3; for horse: equCab2; for pig: susScr11; for mouse: mm10; for zebrafish: danRer10.
Supplementary_files_format_and_content: bigWig files represent the coverage of the ATAC-seq signal.

organism: Canis lupus familiaris

source_name: Melanoma cell line

growth_protocol: The dog cell lines Dog-IrisMel-14205 and Dog-OralMel-18249 were established by Aline Primot , and were derived from an uveal melanoma from a Beagle crossed dog and an oral melanoma from the palate from a Shih-tzu, respectively. Cells were cultured at 37°C with 5% CO2 in Ham's F-12 Nutrient Mixture medium (Thermo Fisher Scientific) supplemented with 10% FBS (Invitrogen) and 1% penicillin/streptomycin (Thermo Fisher Scientific).

extract_protocol:
Cells were washed, trypsinised, spun down at 1000 RPM for 5 min, medium was removed and the cells were resuspended in 1 mL medium. Cells were counted and experiments were only continued when a viability of above 90% was observed. 50,000 cells were used per sample. OmniATAC-seq was performed as described previously in Corces et al., 2017; and ATAC-seq as described in Buenrostro et al., 2015.
Library prep was performed as described before in Corces et al., 2017 for OmniATAC-seq samplas; and as in Buenrostro et al., 2013 for ATAC-seq samples.

treatment_protocol: For the knock-down data: SOX10, TFAP2A and the control knockdown was performed in MM001 using respectively a SMARTpool of four siRNAs against, respectively, SOX10 (SMARTpool: ON-TARGETplus SOX10 siRNA, number L017192-00-0005, Dharmacon), TFAP2A (SMARTpool: ON-TARGETplus TFAP2A siRNA, number L-006348-02-0005, Dharmacon) and a negative control pool (ON-TARGETplus non-targeting pool, number D-001810-10-05, Dharmacon) at a concentration of 20 nM for SOX10-KD, and 40 nM for TFAP2A-KD and the control using as medium Opti-MEM (Thermo Fisher Scientific) and omitting antibiotics. The cells were incubated for 72 h before processing.

tag: tissue type

value: Oral melanoma from the palate, Shih-tzu breed

tag: condition

value: Baseline


title: OmniATAC-seq_Dog_Dog-IrisMel-14205_BL

value: Uveal melanoma, Beagle Crossed dog


title: YHR* rep3

status: Public on Apr 20 2020

city: University Park

first: Nabeel

last: Ahmed

state: PENNSYLVANIA

address: 407 Chemistry Building

department: The Huck Institutes of the Life Sciences

institute: Pennsylvania State University

description: mutant yeast

data_processing:
Library strategy: Ribosome Profiling
The raw reads were first trimmed of their 3′ adapter sequence CTGTAGGCACCATCAATTCGTATGCCGTCTTCTGCTTG using cutadapt v1.14
The reads were also subject to a quality filter of at least 20 during the cutadapt run.
Preprocessed reads were first mapped to a set of ribosomal RNA sequences using Bowtie2  and then subsequently the unmapped reads were mapped to the rest of S. cerevisiae reference genome sacCer3 using Tophat2. For each mutant strain, the sequence at the mutated nucleotide positions was altered in the reference genome for accurate mapping of reads
Gene annotations were obtained from Saccharomyces Genome Database (http://www.yeastgenome.org/) on October 30, 2014.
Custom python scripts were implemented for all downstream analyses. Mapped reads were first quantified by their 5′ ends on individual gene transcripts and A-site positions were assigned according to Table 1 in Ahmed et.al., Scientific Reports (2019).  To maintain the accuracy of read assignment, transcripts in which multiple mapped reads constitute more than 0.1% of the reads mapped to the CDS region were not considered in the analysis.
Genome_build: sacCer3
Supplementary_files_format_and_content: wig files containing coverage at chromosome position determined from 5' end of the reads

organism: Saccharomyces cerevisiae

source_name: yeast

growth_protocol: 200 ml of yeast cells grown in YPD, at 30°C to an optical density (OD600) of 0.5

extract_protocol:
Thawed cell lysates were cleared by centrifugation (20,000xg, 5 min, 4°C) and digested by RNase I (AM2295, Ambion; 125 U/1mg nucleic acid) for 1 hr (25°C, 650 rpm) to obtain ribosome footprints. The reaction was stopped by adding 10 µl SUPERase-In (AM2696, Ambion). The digested lysate was loaded onto 10 50% (w/v) sucrose gradients and centrifuged at 35,000 rpm, 4°C for 2.5 hrs. Gradients were fractionated and monosome fractions were collected, pooled and used for RNA purification by hot acid-phenol extraction. 5 µg of purified RNA was depleted for rRNA using the Ribo-Zero Gold for Yeast kit (MRZY1306, Illumina).
Samples were heated at 80°C for 2 min and  50 μg of RNA  was loaded onto a 15% TBE-Urea polyacrylamide gels (Invitrogen) in 1x TBE (Ambion) and run for 65 min at 200 V. Gels were stained for 20 min with SYBR gold (Invitrogen). To recover ribosomal footprints, the gel pieces were excised that contained RNA fragments with a size between 25 and 33 nt. Gel pieces were placed into 0.5 mL gel breaker tubes, nested into a 1.5 mL tube and centrifuged for 3 min at 20,000xg. 0.5 mL 10 mM Tris-HCl pH 7.0 was added and tubes were incubated at 70°C for 10 min with maximal shaking in an Eppendorf thermomixer. Gel pieces were removed using a Spin-X cellulose acetate column (Fisher) and the flow through was transferred to a new tube. 55 μl 3 M NaOAc pH 5.5, 2 μl glycoblue and 0.55 mL isopropanol were added. After mixing, tubes were frozen at −80°C for 1 hr to 16 hr. Samples were centrifuged for 30 min at 20,000xg and 4°C and pellets were washed with ice-cold 80% ethanol and resuspended in 15 μl of 10 mM Tris-HCl pH 7.0. For dephosphorylation, 2 μl 10x T4 polynucleotide kinase buffer without ATP (NEB), 1 μl murine RNase inhibitor and 2 μl T4 polynucleotide kinase (NEB) were added to each sample. Samples were incubated at 37°C for 1 hr before heat inactivation of the enzyme for 10 min at 75°C and precipitation of nucleic acids by adding 0.5 mL 10 mM Tris-HCl pH 7.0, 55 μl 3 M NaOAc pH 5.5, 2 μl glycoblue and 0.55 mL isopropanol and incubating for 1 hr to 16 hr at −80°CSamples were centrifuged for 30 min at 20,000xg and 4°C, pellets were washed with ice-cold 80% ethanol and resuspended in 6-11 μl of 10 mM Tris-HCl pH 7.0. For linker ligation, a maximum of 5 pmol RNA in 5 μl were denatured for 2 min at 80°C before 8 μl 50% sterile filtered PEG MW 8000, 2 μl DMSO, 2 μl 10x T4 RNA Ligase 2 buffer (NEB), 1 μl murine RNase inhibitor, 1 μl 1 μg/μl linker L1 and 1 μl truncated T4 RNA Ligase 2 (NEB) were added and incubated for 2.5 hr at 37°C or 23°C. Nucleic acids were precipitated as described before and resuspended in 6 μl 10 mM Tris-HCl pH 7.0. Samples were run on a 10% TBE-Urea polyacrylamide gel (Invitrogen) in 1x TBE (Ambion) for 50 min at 200 V. Gels were stained for 20 min with SYBR gold and desired gel pieces were excised and RNA was extracted as described before. For reverse transcription, RNA was resuspended in 10 μl 10 mM Tris-HCl pH 7.0 and 1 μl 10 mM dNTP (NEB), 1 μl 25 linker L1’L2′ and 1.5 μl DEPC H20 were added to each sample. Samples were incubated at 65°C for 5 min followed by addition of 4 μl 5x FSB buffer (Invitrogen), 1 μl murine RNase inhibitor, 1 μl 0.1 M DTT (Invitrogen) and 1 μl Superscript III (Invitrogen). Samples were incubated at 50°C for 30 min and afterward 2.3 μl 1 N NaOH was added to hydrolyze RNA and samples were further incubated at 95°C for 15 min. Samples were run on a 10% TBE-Urea polyacrylamide gel for 70 min at 200 V. Gels were stained as described before and desired bands were excised and nucleic acids were extracted as mentioned earlier but using Tris-HCl pH 8.0 and precipitating nucleic acids by adding 32 μl 5 M NaCl, 1 μl 0.5 M EDTA, 2 μl glycoblue and 0.55 mL isopropanol. For circularization, DNA was resuspended in 15 μl 10 mM Tris-HCl pH 8.0 and 2 μl 10x CircLigase buffer (EPICENTRE), 1 μl 1 mM ATP, 1 μl 50 mM MnCl2 and 1 μl CircLigase™ (EPICENTRE) were added. Samples were incubated at 60°C for 1 hr. Addition of 1 μl CircLigase™ was repeated and samples were incubated for another hour at 60°C. Afterward, the enzyme was inactivated by incubating 10 min at 80°C. 5 μl of circularized DNA was used for PCR amplification. Therefore, 16.7 μl 5x HF buffer, 1.7 μl 10 mM dNTPs, 0.4 μl 100 μM PCR primer L1’, 0.4 μl 100 μM barcoding primer, 59.2 μl DEPC H20 and 0.8 μl HF Phusion (NEB) were added. 17 μl PCR mix were aliquoted to 4 separate PCR tubes and the following PCR reaction cycles were run: 1.) 98°C, 30 s; 2.) 98°C, 10 s; 3.) 60°C, 10 s; 4.) 72°C, 5 s. Steps 2 through 4 were repeated ten times and one tube was removed after cycles 7, 8, 9, 10. Samples were run on a 8% TBE polyacrylamide gel (Invitrogen) in 1x TBE (Ambion) for 45 min at 180 V. Gels were stained as mentioned before and desired bands were excised and DNA was extracted as described before. After a quality control step using a high sensitivity bioanalyzer chip (Agilent), samples were sequenced on a HiSeq 2000 (Illumina)

treatment_protocol: Cells were rapidly filtered (All-Glass Filter 90mm, Millipore), flash frozen in liquid nitrogen, mixed with 600 µL frozen lysis buffer (20 mM Tris-HCl pH 8.0, 140 mM KCl, 6 mM MgCl2, 0.1% NP-40, 0.1 mg/ml CHX, 1 mM PMSF, 2x Complete EDTA-free protease inhibitors (5056489001, Roche), 0.02 U/ml DNase I (4716728001, Roche), 20 mg/mL leupeptin, 20 mg/mL aprotinin, 10 mg/mL E-64, 40 mg/mL bestatin) and pulverized by mixer milling (2 min, 30 Hz, MM400, Retsch).

tag: strain

value: p.L128N(AAC), p.E149K(AAA), p.V250Y(UAC) and p.V338H(CAC) mutations in YHR179W


title: OVCAR3-IFN

status: Public on Apr 09 2020

city: Montréal

first: Claude

last: Perreault

state: Québec

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute for Research in Immunology and Cancer

data_processing:
Basecalls performed using Illumina RTA 2.4.11.
Samples demultiplexed using bcl2fastq 2.20 allowing 1 mismatch in the index.
Sequenced reads were trimmed for sequencing adapters and low quality extrimities and mapped to the reference genome using STAR version 2.5.1b
Readcounts were computed by STAR during sequence alignment based on annotations from Ensembl version 88
Expression values were also computed using RSEM version 1.2.28 based on Ensembl version 88 transcript annotations
Genome_build: GRCh38
Supplementary_files_format_and_content: Readcounts: Tab-delimited file where columns are Ensembl gene identifier, counts for unstranded RNA, counts for 1st strand aligned, counts for 2nd strand aligned
Supplementary_files_format_and_content: RSEM: Tab-delimited file where columns correspond to a standard RSEM output for gene values (first line identifies the column)

molecule: polyA RNA

source_name: ovary

growth_protocol: OVCAR3 cells were cultured in RPMI medium 1640 (Gibco, A10491) supplemented with 10% FBS (Gibco, 12483) and 1% Penicillin Streptomycin (Gibco, 15140).

extract_protocol:
Total RNA was isolated using TRIzol (Invitrogen) as recommended by the manufacturer.
cDNA libraries were prepared from polyA-enriched mRNA using the KAPA Stranded mRNA-Seq Kit and were further amplified for sequencing.

treatment_protocol: 12.5 ng/mL of IFN-g (Life, PHC4031) was added to the culture medium for 72 hours

tag: disease

value: ovarian serous adenocarcinoma

tag: tissue

value: Ovary

value: high-grade serous ovarian cancer (HGSC) cell line


title: Ikbkap_CNS_CKO_2

status: Public on Apr 30 2020

city: Baltimore

first: Supriyo

last: De

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

institute: NIA-IRP, NIH

hyb_protocol: Standard Illumina protocol. In short, a total of 0.75 μg of biotin-labeled cRNA was hybridized at 58°C for 16 hours to Illumina's Sentrix Mouse Red-8 v3 Expression Bead Chips (Illumina, San Diego, CA). Each BeadChip has more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.

scan_protocol: Arrays were scanned at a resolution of 0.8um using the Illumina Beadstation 500 X

data_processing: Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.  Complete data including detection scores is supplied.

source_name: CNS_Ikbkap CKO_2

label_protocol: Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5 μg of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.

extract_protocol: Total RNA was extracted using the Rneasy Plus Mini kit (QIAGEN, Valencia, CA) as per the manufacturers instructions. RNA quality and quantity was assessed using the RNA 6000 Nano Chip on an Agilent Bioanalyzer (Agilent, Wilmington, DE).

treatment_protocol: Examining the major genetic pathways that require IKAP are hampered by that Ikbkap null mice are embryonic lethal (E10), but can be examined by genome-wide transcriptome analysis of the dorsal root ganglia from Wnt1-Cre;IkbkapLoxP/LoxP  E17.5 fetuses (Ikbkap is deleted from the neural crest), or in the brains in adult Tuba1a-Cre;IkbkapLoxP/LoxP   mice. Deletion of Ikbkap from the neural crest lineage induces a caspase-3, p53-dependent cell loss with the pups dying within postnatal day 1, while mice with a deletion of Ikbkap in postmitotic neurons of the PNS and CNS live on average 6 months. These lines were obtained by crossing mice homozygous for a floxed allele of Ikbkap (exon 4) mice to mice that are heterozygous for this floxed allele, and either (1) a Wnt1-Cre transgene or (2) a Tuba1a-Cre transgene. Dorsal root ganglia were removed from E17-18 Wnt1-Cre;IkbkapLoxP/LoxP embryos and brains (cerebellum and brainstem) removed from adult Tuba1a-cre;IkbkapLoxP/LoxP  mice and stored at -80 until RNA was isolated.

tag: genotype/variation

value: CKO

value: CNS

tag: region

value: Cerebellum and brainstem

tag: age

value: adult


title: Ikbkap_CNS_CKO_1

source_name: CNS_Ikbkap CKO_1


title: Ikbkap_CNS_CKO_3

source_name: CNS_Ikbkap CKO_3


title: Sample 4: POL1 Low/LGR5 negative

value: LGR5 negative


title: YHR* rep4

description: mutant yeast


title: UBD_3: CSB UBDmut. Rep3

status: Public on Mar 16 2020

description: Patient-derived cell lines with mutations in CSB (CS1AN) stable expressing CSB with UBD mutant which is first described in Anindya et al. 2010 Mol. Cell. A highly-conserved C-terminal ubiquitin-binding domain (UBD) plays roles in promoting survival/viability and recovery of transcription after DNA damage and oxidative stress

data_processing:
Raw image data file from high-throughput sequencing(like Illumina ) was transformed to Sequenced Reads (called Raw Data or Raw Reads) by CASAVA base recognition (Base Calling). Raw Data is stored in FASTQ(fq) format files, which contain reads sequence and corresponding base quality.FASTQ format described by four lines:@HWI-ST1276:71:C1162ACXX:1:1101:1208:2458 1:N:0:CGATGTNAAGAACACGTTCGGTCACCTCAGCACACTTGTGAATGTCATGGGATCCAT+#55???BBBBB?BA@DEEFFCFFHHFFCFFHHHHHHHFAE0ECFFD/AEHHLine 1 begins with a '@' character and is followed by a sequence identifier and an optional description (like a FASTA title line).Line 2 is the raw sequence letters.Line 3 begins with a '+' character and is optionally followed by the same sequence identifier (and any description) again.Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence.The details of Sequencing identifier of Illumina are as follows:(1) HWI-ST1276:71 HWI-ST1276,Instrument – unique identifier of the sequencer; 71,run number – Run number on instrument(2) C1162ACXX:1:1101:1208:2458 means the coordinate of read on C1162ACXX(Flowcell ID)flowcell,line 1, 1101 tile is(x=1208,y=2458)(3) 1:N:0:CGATGT the first number is 1 or 2, 1 means single reads or the first read of paired ends, 2 means the second of paired ends; the second letter means whether reads is adjusted(Y means yes, N means no); the third number represent the number of Control Bits in sequence; six bases on the fourth place is Illumina index sequence.
For RNA-seq technology, sequencing error rate distribution has two features. Error rate grows with sequenced reads extension for the consumption of sequencing reagent. The phenomenon is common in the Illumina high-throughput sequencing platform (Erlich Y, Mitra PP et al.2008; Jiang L, Schlesinger F et al.2011.). The first six bases have a relatively high error rate. The reason is incomplete binding of the random hex-primers and RNA template in cDNA synthesis (Jiang et al.). In general, a single base error rate should be lower than 1%.
GC-content is used to detect potential AT/GC separation.
The Sequenced Reads/raw reads often contain low quality reads or reads with adaptors. In order to avoid this, it's necessary to filter the raw reads under conditions to get the clean reads. Raw reads filtering conditions are as follows:(1) Remove reads containing adaptors;(2) Remove reads containing N > 10% (N represents base that could not be determined);(3) The Qscore (Quality value) of over 50% bases of the read is <= 5.RNA-seq Adapter information:NEBNext® UltraTM RNA Library Prep KitRNA 5' Adapter(5'Adaptor)5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTRNA 3' Adapter(3'Adaptor,The underlined 6bp bases is Index)5' GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
Genome_build: hg19
Supplementary_files_format_and_content: MS Excel format containing raw counts

source_name: Cockayne Syndrome Patient Derived Fibroblasts

growth_protocol: Cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified chamber under 5% CO2 at 37 °C.

extract_protocol:
Cell are harvested with Buffer RLT. RNA was harvested using Rneasy mini kit extraction Kit (Invitrogen cat no: 74104)
Briefly, mRNA from Eukaryote organisms is purified from total RNA using poly-T oligo-attached magnetic beads (For prokaryotes, mRNA was purified through the removal of rRNA by using kit). The mRNA is first fragmented randomly by addition of fragmentation buffer. According to demand of customer, we have NEB library and strand specific library to choose.

value: CS1AN Cockayne Syndrome Patient Derived Human Fibroblasts

value: pCSB-UBD-GFP (Ubiquitin Binding Domain (UBD) mutant L1427L1428 to G1427G1428 (UBDmut)) were introduced into pCSB-GFP) expressing stable cell line


title: GFP_1: CSB m/m rep1

description: Patient-derived cell lines with mutations in CSB (CS1AN) first described in Troelstra et al. Cell. 1992.

value: pEGFP-C1 expressing stable cell lines


title: UBD_2: CSB UBDmut. Rep2


title: GFP_3: CSB m/m rep3


title: GFP_4: CSB m/m rep4


title: Ikbkap_CNS_WT_1

source_name: CNS_Ikbkap WT_1

value: wild type


title: Ikbkap_CNS_WT_4

source_name: CNS_Ikbkap WT_4


title: Ikbkap_CNS_WT_3

source_name: CNS_Ikbkap WT_3


title: CSB_3: CSB WT rep3

description: Patient-derived cell lines with mutations in CSB (CS1AN) stable expressing full lenght CSB. CSB play roles in DNA repair and other aspects of DNA metabolism.

value: pCSB-GFP expressing stable cell lines


title: UBD_1: CSB UBDmut. Rep1


title: Sample 1: POL1 High/LGR5 Positive

value: POL1 High

value: LGR5 Positive


title: Input-for-ChIP-seq

status: Public on Mar 14 2020

first: Ross

last: Hardison

state: PA

address: 303 Wartik Lab

description: ChIP-seq (input)

data_processing:
Raw sequencing reads were mapped to hg19 using Bowtie. SAM files were converted to BAM files using SAMtools. For histone marks (H3K27Ac), BAM files were converted to Wiggle using MACS (Zhang et al., Genome Biol., 2008) and subsequently converted to bigWig for visualization. Subsequent peak calling was performed using Sicer (Xu et al., Methods Mol Biol., 2014).
Basecalls using bcl2fastq2 v2.15.0.4, and parameters  --barcode-mismatches 1
Mapping to reference genome hg19 canon with Bowtie 1.0.0 using parameters --chunkmbs 1024 -y -n 2 --best -k 1 --maxbts 800 -l 28 -e 80 --sam-nohead --sam
Wiggle generated with MACS using parameters --nomodel --shiftsize=120--format BAM --gsize 2615371906 --tsize 36 --bw 120 --mfold 12 --wig --space 1
Peaks called with Sicer V1.1 using parameters hg19 1 200 150 0.74 600 0.01
Genome_build: Filter blacklist regions from peaks, and convert to broadpeak format (see UCSC Genome Browser for format specs)
Supplementary_files_format_and_content: bw alignment files

growth_protocol: HAP1 cells (Carette et al., Nature 477, 340-343 (2011)), derived from KBM7 cells, a chronic myelogenous leukemia cell line, were cultured in IMDM with 10% FBS and 1% penicillin/streptomycin at 37°C with 5% CO2 (as described in Carette et al., 2011).

extract_protocol: Chromatin immunoprecipitation (ChIP) was performed as previously described (Hsiung, et al., Genes Dev., 2016), using the H3K27Ac antibody (Active Motif 39685). Briefly, ~20 million HAP1 cells were fixed in 1% formaldehyde in fresh culture medium at room temperature for 10 minutes, followed by quenching in 1M glycine for 5 minutes. Crosslinked cells were lysed for 10 minutes in 1mL cold Cell Lysis Buffer (10mM Tris pH 8.0, 10mM NaCl, and 0.2% NP-40/Igepal), supplied with Protease Inhibitors (Sigma-Aldrich P8340) and phenylmethylsulfonyl fluoride (PMSF). Nuclei were pelleted, resuspended in 1mL room temperature Nuclei Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS), with Protease Inhibitors and PMSF, and were incubated on ice for 20 minutes. The samples were sonicated at 100% amplitude, 30 seconds on/30 seconds off, for 45 minutes, in a bath sonicator (QSonica Q800R3). Sonicated materials were centrifuged, with the supernatant subsequently collected, and diluted with 4mL IP Dilution Buffer (20mM Tris pH 8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS), with Protease Inhibitors and PMSF. 50µL protein A/G agarose beads (Thermo Fisher 15918014, Thermo Fisher 15920010) and 50µg isotype-matched IgG control were added to sonicated chromatin to pre-clear it for >2 hours at 4°C. Beads were then spun down, with 200µL supernatant containing pre-cleared chromatin saved as “input” before immunoprecipitation. The remaining pre-cleared chromatin was split into equal volumes, each incubated with antibody or isotype-matched control (IgG) pre-bound protein A/G beads, and rotated overnight at 4°C. Chromatin-bound beads were washed on ice, once with IP Wash 1 (20mM Tris pH 8, 2mM EDTA, 50mM NaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20mM Tris pH 8, 2mM EDTA, 500mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash 2 (10mM Tris pH 8, 1mM EDTA, 0.25M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate), and twice with TE. Beads were then moved to room temperature, and were eluted twice with a total volume of freshly prepared 200µL Elution Buffer (100mM NaHCO3, 1% SDS). Into each IP and input, 12µL 5M NaCl and 2µL RNase A (10mg/mL, Roche through Sigma 10109169001) were added, and samples were incubated at 65°C overnight. 3µL proteinase K (20mg/mL, Roche through Sigma 3115879) was then added, for an additional 2 hours at 65°C. DNA was column cleaned up using a QIAquick PCR Purification Kit (QIAGEN 28106). For ChIP-sequencing, library construction was performed using Illumina’s TruSeq ChIP sample preparation kit (Illumina IP-202-1012), followed by size selection using SPRIselect beads (Beckman Coulter, B23318). Libraries were quality checked, quantified prior to 1 x 50 bp sequencing on the Illumina NextSeq 500.

treatment_protocol: HAP1 cells were co-transfected with the two components of the Sleeping Beauty transposon system: pSA-MCS-2kb, the transposon vector with the 2kb insert containing a CTCF binding site and the TSS of PARL gene, and the transposase vector, pCMV(CAT)T7-SB100. Clonal lines were established following transposon genome editing. Hi-C was then performed for two clones (Clone 21 and Clone 25) with the highest numbers of insertions. Where indicated, CRISPR editing using a gRNA targeting the CTCF binding site within ectopic insertions was performed, and Capture-C was used to characterize effects upon CTCF binding site disruption.

value: KBM7-derived near haploid cells


title: CSB_2: CSB WT rep2


title: ChIP-seq-H3K27Ac-Clone25

description:
ChIP-seq
ChIP_cpm_bw.tar


title: CSB_4: CSB WT rep4


title: GFP_2: CSB m/m rep2


title: CSB_1: CSB WT rep1


title: Ikbkap_CNS_CKO_4

source_name: CNS_Ikbkap CKO_4


title: ChIP-seq-H3K27Ac-WT

description:
ChIP-seq
ChIP_cpm_bw.tar


title: UBD_4: CSB UBDmut. Rep4


title: RNA-Seq-Clone25-rep2

description:
rRNA-depleted RNA
C21_DESeq_res
C25_DESeq_res
RNA-Seq_near_insertions.tar

data_processing:
Two biological replicates were performed for each cell line. For differential expression (DE) analysis, reads were mapped to the indexed Ensembl hg19 transcriptome (GRCh37.67) and quantified using Salmon (Patro et al., Nat. Methods, 2017). Transcript-level quantifications were then imported, using tximport (Soneson et al., F1000Res, 2015), for gene-level (EnsDb.Hsapiens.v75) measurements with DESeq2 (Love et al., Genome Biol., 2014). Genes with less than 10 reads were excluded from downstream DE analysis. DESeq2 was performed to identify DE genes between Clone 21 and non-Clone 21, and between Clone 25 and non-Clone 25, at an FDR < 0.01. For RNA-seq analysis for genome tracks visualization, the sequence reads were processed using the ENCODE long RNA-Seq pipeline (https://www.encodeproject.org/pipelines/ENCPL002LPE/). Briefly, raw sequencing reads were initially accessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Mapping to hg19 was performed using STAR (Dobin et al., Bioinformatics, 2013). Resulting bedGraph files were then converted to bigWig format for genome browser visualization with strand specificity.
Genome_build: hg19
Supplementary_files_format_and_content: "C21_DESeq_res" and "C25_DESeq_res" are quantification results using Salmon (Patro et al., Nat. Methods, 2017), also catagorizing whether each gene is within a distance range centered around each insertion site. "RNA-Seq_near_insertions.tar" are extracted bedgraphs from alignment bigwig file for visualization purposes.

extract_protocol: HAP1 cells were washed with PBS, and resuspended in 1mL TRIzol (Thermo Fisher), with 200µL chloroform then added. RNA was extracted using RNeasy Mini Kit (Qiagen). Sequencing libraries were constructed from 500 ng of DNase-treated, total RNA using the ScriptSeqv2 Complete Kit (Illumina BHMR1224). Briefly, the RNA was depleted of rRNA using the Ribo-Zero removal reagents and fragmented. First strand cDNA was then synthesized using a 5’ tagged random hexamer and reverse transcription, followed by annealing of a 5’ tagged, 3’-end blocked terminal-tagged oligo and second strand synthesis. The Di-tagged cDNA fragments were purified, barcoded, and PCR-amplified for 15 cycles. Libraries were quality checked and quantified prior to 2 x 76 bp sequencing on the Illumina NextSeq 500.


title: ChIP-seq-H3K27Ac-Clone21

description:
ChIP-seq
ChIP_cpm_bw.tar


title: Ikbkap_CNS_WT_2

source_name: CNS_Ikbkap WT_2


title: RNA-Seq-WT-rep2


title: RNA-Seq-Clone21-rep1


title: Capture-C-InsertProbe-WT-rep1

description:
Capture-C
Capture-C_MLKL_locus.tar

data_processing:
Library strategy: Capture-C
Two biological replicates for each cell line were performed. Raw reads were processed using published scripts (Davies, et al., Nat. Methods, 2016). Briefly, Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), a wrapper for FastQC and Cutadapt, was used to assess the quality and to trim adapter sequences of raw sequences. Trimmed reads were then merged or interleaved using FLASH. Once concatenated, the reads were in silico DpnII digested, aligned to hg19, and analyzed using CCanalyser3. Interactions were then normalized to total interactions. Bedgraph files from each individual replicate were merged.
Genome_build: hg19
Supplementary_files_format_and_content: For each locus with an insertion, and with a CRISPR editing, where applicable, each .tar has a collection of BedGraph files generated by combining the biological replicates of each sample

extract_protocol: Capture-C was performed as previously described (Hughes et al., Nat. Genet., 2014; Huang et al., Genes Dev., 2017; Davies et al., Nat. Methods, 2016). Briefly, ~10 million cells HAP1 cells were crosslinked in 1% formaldehyde at room temperature for 10 minutes, and then quenched in 1M glycine for 5 minutes, on an orbital shaker. Cells were then pelleted, washed with 1mL cold PBS, transferred to an Eppendorf tube, pelleted, resuspended in 1mL cold Cell Lysis Buffer (10mM Tris pH 8.0, 10mM NaCl, and 0.2% NP-40/Igepal), and incubated on ice for 10 minutes. Nuclei were then pelleted, washed once using NEBuffer™ DpnII, pelleted, and resuspended in 500µL NEBuffer™ DpnII. Samples were incubated in 0.3% SDS at 37°C, 950 rpm for 1 hour in a thermomixer, followed by the addition of a final concentration of 1.8% Triton X-100, for 1 hour at 37°C, 950 rpm. After adding 40µL water, 300U DpnII was added for in situ digestion at 37°C, overnight. An additional 300U DpnII was then added, and samples were incubated for 4 more hours at 37°C, followed by 20 minutes at 65°C, 950 rpm. Nuclei were pelleted, resuspended in a total volume of 1.2mL with 4000U T4 DNA Ligase (NEB), and incubated at 16°C for 4 hours and then at room temperature for 30 minutes. 20µL of 20mg/mL Proteinase K and 120µL of 10% SDS were added, and samples were incubated at 65°C overnight to reverse crosslinking. An additional 10µL proteinase K was added to each sample for 2 more hours at 55°C. 2µL DNase-free RNAase was added to each sample, followed by incubation at 37°C for 30 minutes. DNA was then extracted with phenol-chloroform. DNA was sonicated to 200-300bp in a bath sonicator (QSonica Q800R3), followed by beads clean-up. Sequencing libraries were subsequently prepared similarly as described in "Insertion-site-mapping” samples: End Repair, dA-Tailing, adaptor ligation, 6 cycles of indexing PCR, and 6 cycles of amplification using P5/P7. After sequencing library preparation, two rounds of target enrichment/capture were carried out using biotinylated hybridization oligos similarly as described in "Insertion-site-mapping” samples. Pulldown enrichment was confirmed using qPCR. Target enriched libraries were quality checked and quantified as in situ Hi-C samples prior to 2 x 75 bp sequencing on the Illumina NextSeq 500.


title: Capture-C-InsertProbe-WT-rep2

description:
Capture-C
Capture-C_MLKL_locus.tar


title: Capture-C-InsertProbe-Clone25-rep2

description:
Capture-C
Capture-C_MLKL_locus.tar


title: Capture-C-InsertProbe-Clone25-rep1

description:
Capture-C
Capture-C_MLKL_locus.tar


title: Capture-C-WT-rep1

description:
Capture-C
Capture-C_chr2_13M_locus.tar
Capture-C_chr6_75M_locus.tar
Capture-C_chr2_232M_locus.tar
Capture-C_MLKL_locus.tar
Capture-C_chr2_13M_locus_FullSet.tar
Capture-C_chr6_75M_locus_FullSet.tar
Capture-C_DirectionalityIndex_InsertionLoci.tar


title: Capture-C-Clone21-rep2

description:
Capture-C
Capture-C_chr2_13M_locus.tar
Capture-C_chr6_75M_locus.tar
Capture-C_chr2_13M_locus_FullSet.tar
Capture-C_chr6_75M_locus_FullSet.tar
Capture-C_DirectionalityIndex_InsertionLoci.tar


title: Capture-C-Clone21-CTCF-CRISPR-rep1


title: Capture-C-WT-rep2


title: Capture-C-Clone25-rep1

description:
Capture-C
Capture-C_chr2_232M_locus.tar
Capture-C_MLKL_locus.tar
Capture-C_chr2_13M_locus_FullSet.tar
Capture-C_chr6_75M_locus_FullSet.tar
Capture-C_DirectionalityIndex_InsertionLoci.tar


title: Capture-C-Clone21-CTCF-CRISPR-rep2


title: Hi-C-WT-rep1-deeper-seq

description:
Hi-C
hap1_wt_hic_20000_ord.bed
hap1_wt_hic_20000_iced.matrix
hap1_wt_endo.matrix
Hi-C_Matrices_near_C21_insertions.tar
Hi-C_Matrices_near_C25_insertions.tar
Hi-C_Matrices_LocusFromNoInsertionChromosome.tar
eigen_chr6.tar
Hi-C_Matrices_14M_around_C21S4_VisualizeCompart.tar
Hi-C_Matrices_14Mproximal_40Mdistal_C21S4_VisualCompart.tar
Hi-C_Matrices_2kb_endogenous_CRISPR_genotyped.tar
Hi-C_Eigen_Insertion_or_CRISPRed_Chromosomes.tar
hap1_wt_hic_allValidPairs.hic

data_processing:
Two replicates of each sample, WT, Clone 21 and Clone 25, underwent a pilot sequencing run, generating ~30 million raw reads for each replicate. Reads from each replicate were then mapped to hg19 using Bowtie 2. Detection and filtering of valid interaction pairs, assignment to restriction fragment and binning, and interaction matrix balancing with iterative correction and eigenvector decomposition (ICE) (Imakaev, et al., Nat. Methods, 2012) were all performed using the HiC-Pro pipeline (Servant, et al., Genome Biol. 2015). With each replicate of a given sample having highly reproducible metrics: >60% valid interaction pairs-raw read pairs ratio, <20% trans interaction, <8% dangling pairs, and contact ranges of interaction pairs, two replicates of the sample were subsequently pooled for further analysis. Deeper sequencing was subsequently performed to yield ~250 million raw reads for each sample, generating ~160 million valid interaction pairs per sample, after HiC-Pro processing. ICE balanced interaction matrices were binned at 20kb resolution, and used for downstream analyses, unless otherwise specified. Genome-wide Hi-C contacts Pearson correlations between two samples were calculated on non-empty bins shared between two samples.                             To generate heatmaps for each insertion site, ICE balanced matrices for the clone with the insertion, as well as for WT and the other clone without the insertion at this position, were extracted with the insertion bin in the middle and with 150 bins (3 million bps) on either side of it. These extracted matrices, adjusted for minor coverage differences, were plotted using lib5C (Gilgenast, et al., Cell Syst., 2019) in linear scale using color scheme “red8” from matplotlib (Hunter, et al., Computing in Science Engineering, 2007), highlighting a megabase-scale region centering around each insertion.                         Insulation score computations were implemented mechanistically similar to those in (Crane et al., Nature, 2015). Given an ICE balanced interaction matrix with the insertion bin at the center, averaged interactions within a sliding window of 10 bins by 10 bins (15 bins by 15 bins when the insert was at a pre-established boundary or at the endogenous boundary where the 2 kb test DNA was chosen), were recorded for each bin along the diagonal of interaction matrices. The insulation score for each diagonal bin was then normalized to all insulation scores across its nearby 240 bins (4.8 Mb chromosomal region), with the log2 ratio subsequently calculated. Positive log2 values indicate relatively enriched interactions, whereas negative log2 values suggest relatively depleted interactions, with local minima marking domain boundaries. Shared domain boundaries genome-wide among WT and insertion clones were identified similarly: averaged interactions within a sliding window of 10 bins by 10 bins, log2 after normalized to all scores chromosome-wide, and local minima determined, with a minimum domain size of 4 bins (80 kb).
Genome_build: hg19
Supplementary_files_format_and_content: hap1_wt_hic_20000_ord.bed: coordinates to bin # (each bin 20 kb) lookup table; hap1_wt/clone21/clone25_hic_20000_iced.matrix: genome-wide sparse matrix; hap1_wt_endo.matrix: extracted around the locus where the 2kb test DNA was derived from. Eigen_chr6.tar: eigenvalues of chr6, binned at 50 kb, where Clone 21 insertion site #4 resulting in a likely compartment change occurred.

extract_protocol: In situ Hi-C was performed as previously described (Rao et al., Cell, 2014; Nora et al., Cell, 2017; Huang et al., Genes Dev. 2017.). Briefly, ~10 million HAP1 cells, WT, Clone 21 and Clone 25, were crosslinked in 2% formaldehyde at room temperature for 10 minutes, and then quenched in 0.125M glycine for 5 minutes, on an orbital shaker. Cells were then transferred from a culture flask to a 15mL tube, pelleted, washed with 1mL cold PBS, transferred to an Eppendorf tube, pelleted, resuspended in 1mL cold Cell Lysis Buffer (10mM Tris pH 8.0, 10mM NaCl, and 0.2% NP-40/Igepal), and incubated on ice for 10 minutes. Nuclei were then pelleted, washed once using 800µL NEBuffer™ DpnII, pelleted, and resuspended in 500µL NEBuffer™ DpnII. A final concentration of 0.3% SDS was added, and samples were incubated at 37°C, 950 rpm for 1 hour in a thermomixer. A final concentration of 1.8% Triton X-100 was then added to each sample, which was subsequently incubated at 37°C, 950 rpm for 1 hour. 40µL nuclease-free water, as well as 300U DpnII (NEB R0543M), was added, and samples were digested at 37°C, at 950 rpm overnight in a thermomixer. Another 300U DpnII were added for an additional 4 hours of digestion with mixing. The samples were then incubated at 65°C, 950 rpm for 20 minutes. Nuclei were then pelleted, resuspended in 1X NEBuffer™ 2, with biotin-14-dATP, dTTP, dCTP and dGTP and DNA polymerase I, Large (Klenow) Fragment (NEB M0210), and incubated at 37°C, 950 rpm in a thermomixer for 90 minutes. Ligation reaction was subsequently carried out in a total volume of 1.2mL with 4000U T4 DNA Ligase (NEB) at 16°C for 4 hours, followed by 30 minutes at room temperature. 20µL of 20mg/mL Proteinase K and 120µL of 10% SDS were then added to each sample, followed by crosslinking reversal at 65°C overnight. An additional 10µL proteinase K was added to each sample, which was incubated at 55°C for 2 hours. 2µL DNase-free RNAase was added to each sample, followed by incubation at 37°C for 30 minutes. Phenol-chloroform extraction was performed to purify DNA. DNA was sonicated to 200-300bp in a bath sonicator (QSonica Q800R3), followed by beads clean-up. Biotinylated nucleotides-filled ligation junctions were pulled down with 50µL Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher 65001). Sequencing libraries were subsequently prepared: End Repair, dA-Tailing, adaptor ligation, followed by 6 cycles of indexing PCR. The quality and size (average 429 bp) of each library was evaluated using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems, KK4835). Libraries were then pooled and sequenced in paired-end mode on the NextSeq 500 to generate 2 x 75 bp reads using Illumina-supplied kits as appropriate.


title: Hi-C-Clone21-rep1-deeper-seq

description:
Hi-C
hap1_clone21_hic_20000_iced.matrix
Hi-C_Matrices_near_C21_insertions.tar
Hi-C_Matrices_near_C25_insertions.tar
Hi-C_Matrices_LocusFromNoInsertionChromosome.tar
eigen_chr6.tar
Hi-C_Matrices_14M_around_C21S4_VisualizeCompart.tar
Hi-C_Matrices_14Mproximal_40Mdistal_C21S4_VisualCompart.tar
Hi-C_Eigen_Insertion_or_CRISPRed_Chromosomes.tar
hap1_clone21_hic_allValidPairs.hic


title: RNA-Seq-Clone25-rep1


title: Hi-C-WT-rep2-deeper-seq


title: Hi-C-Clone21-rep2-deeper-seq


title: Hi-C-Clone25-rep2-deeper-seq

description:
Hi-C
hap1_clone25_hic_20000_iced.matrix
Hi-C_Matrices_near_C21_insertions.tar
Hi-C_Matrices_near_C25_insertions.tar
Hi-C_Matrices_LocusFromNoInsertionChromosome.tar
eigen_chr6.tar
Hi-C_Matrices_14M_around_C21S4_VisualizeCompart.tar
Hi-C_Matrices_14Mproximal_40Mdistal_C21S4_VisualCompart.tar
Hi-C_Eigen_Insertion_or_CRISPRed_Chromosomes.tar
hap1_clone25_hic_allValidPairs.hic


title: Capture-C-Clone21-rep1


title: RNA-Seq-Clone21-rep2


title: Hi-C-Clone21-rep2-pilot-seq


title: Hi-C-Clone25-rep1-pilot-seq


title: Hi-C-Clone21-rep1-pilot-seq


title: Hi-C-WT-rep2-pilot-seq


title: Hi-C-Clone25-rep2-pilot-seq


title: RNA-Seq-WT-rep1


title: Capture-C-Clone25-rep2


title: IR 14D rep 2

status: Public on Mar 04 2020

city: Utrecht

first: Eva

last: van Rooij

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

data_processing:
Reads were mapped with BWA-ALN. Reads that mapped equally well to multiple loci in the reference genome were excluded
The first read of each readpair contains the cell barcode of the CELseq2 protocol and the UMI
The second read of each readpair was mapped to the reference genome  (barcode used in library are depicted in the spreadsheet "Barcodes used" (available on the series record).
Per gene, all mapped reads containing the same cellular barcode and a different UMI in the first read were aggregated to obtain final readcounts
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Readcounts of genes in cells are present in a matrix format, with rows being genes and columns being cells. Readcount tables are in .tsv format

source_name: cardiac_IR 14D rep 2

growth_protocol: Mouse were kept unde normal housing conditions

extract_protocol:
Single cardiac cells were collected according to a protocol of Gladka et al. (Circulation, 2018). cDNA preparation was performed using the SORT-seq protocol (Muraro et al., Cell Systems, 2016).  cDNA libraries were obtained by dispersion of the RT enzyme and second strand mixes with the Nanodrop II liquid handling platform (GC biotech).
Illumina sequencing libraries were prepared using the TruSeq small RNA primers (Illumina).

treatment_protocol: All mice underwent either ischemia-reperfusion (IR) or a sham surgery. IR was performed by a 1-hour temporal ligation of the left anterior descending (LAD) artery. Cardiac tissue was collected at several time points post surgery.

value: Cardiac (left ventricle - infarcted region)

value: IR

tag: time (days)

value: 8 weeks

tag: Sex

value: Male


title: IR 14D rep 1

source_name: cardiac_IR 14D rep 1


title: Hi-C-Clone25-rep1-deeper-seq


title: IR 3D rep 5

source_name: cardiac_IR 3D rep 5


title: IR 14D rep 3

source_name: cardiac_IR 14D rep 3


title: IR 3D rep 4

source_name: cardiac_IR 3D rep 4


title: IR 3D rep 6

source_name: cardiac_IR 3D rep 6


title: IR 3D rep 1

source_name: cardiac_IR 3D rep 1


title: IR 3D rep 3

source_name: cardiac_IR 3D rep 3


title: Hi-C-WT-rep1-pilot-seq


title: IR 3D rep 2

source_name: cardiac_IR 3D rep 2


title: IR 3D rep 7

source_name: cardiac_IR 3D rep 7


title: IR 1D rep 7

source_name: cardiac_IR 1D rep 7


title: Sham 14D rep 7

source_name: cardiac_Sham 14D rep 7

value: sham


title: IR 1D rep 4

source_name: cardiac_IR 1D rep 4


title: IR 1D rep 8

source_name: cardiac_IR 1D rep 8


title: IR 1D rep 3

source_name: cardiac_IR 1D rep 3


title: Sham 14D rep 8

source_name: cardiac_Sham 14D rep 8


title: IR 1D rep 5

source_name: cardiac_IR 1D rep 5


title: IR 1D rep 2

source_name: cardiac_IR 1D rep 2


title: IR 1D rep 9

source_name: cardiac_IR 1D rep 9


title: IR 1D rep 1

source_name: cardiac_IR 1D rep 1


title: Sham 14D rep 4

source_name: cardiac_Sham 14D rep 4


title: Sham 14D rep 5

source_name: cardiac_Sham 14D rep 5


title: Sham 14D rep 3

source_name: cardiac_Sham 14D rep 3


title: Sham 14D rep 6

source_name: cardiac_Sham 14D rep 6


title: Sham 1D rep 4

source_name: cardiac_Sham 1D rep 4


title: Sham 14D rep 1

source_name: cardiac_Sham 14D rep 1


title: Sham 1D rep 2

source_name: cardiac_Sham 1D rep 2


title: Sham 1D rep 1

source_name: cardiac_Sham 1D rep 1


title: Sham 1D rep 3

source_name: cardiac_Sham 1D rep 3


title: IR 1D rep 6

source_name: cardiac_IR 1D rep 6


title: CS20_human_embryo_01

status: Public on Feb 16 2020

city: Beijing

first: Tao

last: Huang

state: Beijing

address: Dong Da Jie No.8

country: China

institute: The Fifth Medical Center of Chinese PLA General Hospital

data_processing:
Read 1: biological read
Read 2: technical information read (cell barcode (first 8 nt) and UMI (next 8 nt))
Illumina CASAVA version 1.8 were used to the basecalling.
Raw reads of each cell were first split by specific barcode sequence attached in Read 2. The template switch oligo (TSO) sequence and polyA tail sequence were trimmed for the corresponding Read 1 after UMI information was aligned to it. Subsequently, reads with adapter contaminants or low-quality bases (N > 10%) were discarded. Next, the stripped Read1 sequences were aligned to hg19 human transcriptome (UCSC) using TopHat (version 2.0.12) (Trapnell et al., 2009). Uniquely mapped reads were counted by HTSeq package(Anders et al., 2015) and grouped by the cell-specific barcodes. Duplicated transcripts were removed based on the UMI information for each gene. Finally, for each individual cell, the copy number of transcripts of a given gene was the number of the distinct UMIs of that gene.
Genome_build: hg19
Supplementary_files_format_and_content: Quality_controled_UMI_data_*.txt is the expression matrix.
Annotations_*.txt is the metadata_of_exp_matrix

source_name: human embryonic cells

extract_protocol:
The various tissues (yolk sac, head, lung, skin, and liver) were carefully dissected under the microscope, with the exception of the blood which was collected by suction pipe from the heart directly. After washing in PBS 3 times to remove any residual blood contamination, the yolk sac, head, lung and skin were transferred to pre-warmed digestion medium containing 0.1 g/mL Collagenase I (Sigma, C2674, preheated to 37 °C) in RPMI 1640 medium. The samples were enzymatically digested at 37 °C in a humidified incubator for 20-30 minutes, with the samples being shaken periodically every five minutes until digested to single cell suspension. The liver was first cut into small pieces using scalpels, and then mechanically dissociated using syringes into single cell suspension, before removing the erythrocytes with lysis buffer (BD). The cells were then filtered through a 70 mm cell strainer after neutralizing enzymes. For the child Langerhans cells (LC), the tissue was first incubated in RPMI 1640 with 1.2 U/ml dispase II (Roche, Indianapolis, IN) at 4 °C for 8 h prior to the separation of the epidermal layer. After separation, the epidermal layer was cut into pieces and digested with 0.25% trypsin-EDTA (Gibco) containing 0.25 mg/ml DNase I (Sigma DN25) for 30 min at 37°C in a humified incubator. After that, the epidermal layer was passed through a 70 mm cell strainer by grinding, and then washed with PBS.The following antibodies were used for staining of the embryonic cells: CD45 (BV421, 563879 BD), CD235a (APC-Cy7, 349116 Biolegend) and 7-amino-actinomycin D (7-AAD) (PerCP-Cy5.5, 00699350 eBioscience). LC were isolated with CD45 (BV421, 563879 BD), 7-amino-actinomycin D (7-AAD) (PerCP-Cy5.5, 00699350 eBioscience), CD207 antibodies (PE, 564727 BD) and CD1a antibodies (APC, 559775 Biolegend). Cells were sorted with the Aria 2 Flow Cytometer (BD Bioscience). Data were analyzed with BD FACSDIVA V8.0.1.
Sequencing libraries were constructed following a modified singlecell tagged reverse transcription (STRT) protocol as previously reported(Li et al., 2017; Picelli et al., 2013; Picelli et al., 2014). Initially, Single cells in good condition were picked into the lysis buffer by mouth pipette. The reverse transcription reaction was performed using sample-specific 25 nt oligo dT primer containing 8 nt barcode (TCAGACGTGTGCTCTTCCGATCT-XXXXXXXX-NNNNNNNN-T25, X representing sample-specific barcode whereas N standing for unique molecular identifiers, UMI). After reverse transcription and second-strand cDNA synthesis, the cDNAs were amplified by 16 cycles of PCR. Different labeled cells were then pooled together and purified using Agencount AMPure XP beads. Biotinylated pre-indexed primers were used to further amplify the PCR product by an additional 4 cycles of PCR to introduce biotin tags to the 3’ ends of the amplified cDNAs. Approximately 300 ng cDNA was sonicated into 300 bp fragments with Covaris S2 system and enriched with Dynabeads MyOneTM Streptavidin C1 beads. Libraries were constructed using a Kapa Hyper Prep Kit (Kapa Biosystems) and were then submitted to 150 bp paired-end sequencing on an Illumina HiSeq X ten platform (Novogene).

value: Head, Liver, Blood, Skin, Lung

tag: developmental stage

value: embryonic cells

tag: facs type


title: CS23_human_embryo_01

value: Head, Liver, Skin, Lung

tag: cell type


title: human_child_foreskin_01

source_name: human chlid foreskin cells

value: Foreskin

value: foreskin cells


title: CS11_human_embryo_01

value: Yolk sac, Head


title: CS12_human_embryo_01

value: Yolk sac, Head, Liver


title: CS15_human_embryo_01

value: Head, Liver, Blood


title: CS17_human_embryo_01

value: Yolk sac, Head, Liver, Blood


title: BS58_MHCIIpos_F480hi_AKI

status: Public on Jan 31 2020

city: Planegg

first: Barbara

last: Schraml

address: Großhaderner Strasse 9

country: Germany

institute: LMU Munich

data_processing:
Sequencing reads were aligned to mm10 mouse genome using STAR
Gene expression in transcripts per kilobase million (TPM) was calculated with RSEM
Genome_build: mm10
Supplementary_files_format_and_content: RSEM results (comma separated value file contains TPM values for each sample), reads per gene

source_name: kidney

extract_protocol:
RNA isolation with PicoPure Kit according to protocol. Cells were sorted directly in lysis buffer supplied with the kit
cDNA creation with SMARTseq v4 Kit (Takara), library creation with MicroPlex Library Preparation Kit v2 (Diagenode)

tag: agent

value: cisplatin

tag: pathophysiology

value: cisplatin-induced acute kidney injury (AKI)

value: kidney

value: 10 weeks

value: WT

value: F4/80hi cells

tag: sorting strategy

value: CD45.2+ CD19- Ly6G- Ly6C- MHCII+ CD64+ CD11b+ F4/80hi


title: BS57_MHCIIneg_F480hi_AKI

tag: genotype

value: CD45.2+ CD19- Ly6G- Ly6C- MHCII- CD11b+ F4/80hi


title: ATAC6

status: Public on Feb 06 2020

city: Heidelberg

first: Guenter

last: Raddatz

address: Im Neuenheimer Feld 580

institute: German Center for Cancer Research

data_processing:
Illumina RTA 1.18.64 software used for basecalling.
Preprocessing, read trimming and quality filtering: Trimmomatic 0.39
Read mapping: Bowtie2
Peak calling: MACS2
Calling of count data: htseq.py
Genome_build: mm9
Supplementary_files_format_and_content: Individual peaks, total merged peaks and count tables for all samples

source_name: colon epithel

extract_protocol:
Fresh crypts were extracted and dissociated into single cells. 50,000 cells were sorted and pelleted by centrifugation for 5 min at 500 g and 4°C. Cell pellets were washed once with 1xPBS and pelleted again. Cell pellets were resuspended in 25 µl of lysis buffer and nuclei were pelleted. Supernatant was discarded and nuclei were re-suspended in 50 µl reaction buffer containing 2.5 µl of Tn5 transposase and 25 µl of TD buffer.
Sequencing libraries were prepared using DNA purification (QIAquick minelute columns, Qiagen) and amplification by PCR using Nextera complimentary primers. Paired-end sequencing was performed on an Illumina HiSeq 2000 machine by multiplexing three samples on one lane.

tag: treatment

value: No treatment


title: ATAC7


title: ATAC2

value: DSS-treatment


title: ATAC5


title: Sham 14D rep 2

source_name: cardiac_Sham 14D rep 2

value: Cardiac (left ventricle - infarcted region)

value: sham

value: 8 weeks

value: Male


title: ATAC1


title: BS56_MHCIIpos_F480hi_AKI


title: BS55_MHCIIneg_F480hi_AKI


title: GS300_cDC1

data_processing:
Sequencing reads were aligned to mm10 mouse genome using STAR
Gene expression in transcripts per kilobase million (TPM) was calculated with RSEM
Genome_build: mm10
Supplementary_files_format_and_content: comma separated value file contains TPM values for each sample, *tab files contain read counts.

value: Clec9a-Cre/Cre Rosa-YFP/YFP

value: CD45+ Ly6C- MHCII+ CD11c+ CD64- CCD24+ YFP+


title: BS52_MHCIIpos_F480hi_ctrl

value: control


title: GS292_cDC1


title: BS51_MHCIIpos_F480hi_ctrl


title: BS54_MHCIIpos_F480hi_AKI

value: cisplatin


title: BS53_MHCIIneg_F480hi_AKI

value: cisplatin-induced acute kidney injury (AKI)


title: BS7_cDC1

value: kidney


title: CS13_human_embryo_01

value: Yolk sac, Head, Blood

value: embryonic cells


title: BS4_cDC1

value: 10 weeks


title: CS15_human_embryo_02

value: Yolk sac, Head, Liver, Blood


title: GS284_cDC1


title: BS1_cDC1


title: ATAC4


title: GS299_cDC2

value: CD45+ Ly6C- MHCII+ CD11c+ CD64- CD11b+ YFP+


title: GS283_cDC2


title: GS291_cDC2


title: BS27_cDC2


title: BS21_cDC2


title: BS26_CD11bhi_YFP+

value: CD11bhiF4/80low YFP+

value: CD45+ Ly6C- MHCII+ CD64+ F4/80low CD11bhi YFP+


title: BS20_CD11bhi_YFP+


title: BS9_CD11bhi_YFP+


title: BS25_CD11bhi_YFP-

value: CD11bhiF4/80low YFP-

value: CD45+ Ly6C- MHCII+ CD64+ F4/80low CD11bhi YFP-


title: BS6_CD11bhi_YFP+


title: BS3_CD11bhi_YFP+


title: GS298_F480hi_YFP+

value: F4/80hiCD11blow YFP+

value: CD45+ Ly6C- MHCII+ CD64+ F4/80hi CD11blow YFP+


title: GS282_F480hi_YFP+

value: Clec9a-Cre/Cre Rosa-YFP/YFP


title: GS290_F480hi_YFP+


title: GS297_F480hi_YFP-

value: F4/80hiCD11blow YFP-

value: CD45+ Ly6C- MHCII+ CD64+ F4/80hi CD11blow YFP-


title: GS289_F480hi_YFP-


title: GS281_F480hi_YFP-


title: GS280_cDC1

source_name: spleen

value: spleen

value: Autofluorescence- MHCII+ CD11c+ CD24+ CD205+ YFP+


title: GS287_cDC2

value: Autofluorescence- MHCII+ CD11c+ CD11b+ YFP+


title: GS279_cDC2


title: GS288_cDC1


title: GS285_RPM

value: red pulp macrophages

value: Autofluorescence+ F4/80+ YFP-


title: GS296_cDC1

value: 10 weeks


title: GS295_cDC2


title: GS277_RPM


title: ATAC_seq_wing

status: Public on Jan 27 2020

city: New York City

first: Harmen

middle: J

last: Bussemaker

state: New York

address: 1212 Amsterdam Avenue, MC 2441

department: Biological Sciences

institute: Columbia University

data_processing:
SELEX-seq 16-bp libraries: Fastq files were reverse-complemented and both forward and reverse-complement read merged into one (fastx_reverse_complement -Q33 -z ; FASTX toolkit 0.0.12;Hannon lab). Next, fixed primer sites were trimmed using the following options: fastx_trimmer -Q33 -f 12 -l 41 –z (FASTX toolkit 0.0.12;Hannon lab). Kmer counting and relative affinity estimates were computed using the R package bioconductor.org/packages/SELEX. SELEX-seq 21-bp and 30bp libraries: Sequencing primer sequences were removed as part of the Illumina demultiplexing. For the 21-bp libraries both Hth-F and Hth-R Homothorax orientations are present in the same fastq and can be isolated by using the R package bioconductor.org/packages/SELEX and matching left an right barcode to either CTGTCA-21xN-CCCGGG (Hth-R) or TGACAG-21xN-CCCGGG (Hth-F).
ChIP-Seq: Deposited fastq files contain the merged and demultiplexed files from the four separate NextSeq 500 sequening lanes.
Files were aligned using bowtie-2 to the D. melanogaster genome version dm6.
Aligned sam files were next converted into bam files, sorted and cleared from duplicate reads using the samtools functions view, sort and rmdup. The sorted bam files containing unique reads were indexed (Samtools: index) and converted into bigwig files using the bamCoverage function from the Deeptools suite with parameters -bs 1 -e 125.
Peaks were called using the MACS2 function callpeak with input samples as control and parameters -g dm -q 0.01 --nomodel --extsize 125
ATAC-seq: like ChIP-seq but without removing duplicates.
Genome_build: dm6
Supplementary_files_format_and_content: processed data files contain bigwig coverage tracks for all IP and input samples at 1 bp resolution with reads extended by 125bp. The wild-type Exd-V5 peak file used in the study is provided as a bed file containing the peak summit genome coordinates. No processed data are available for SELEX-seq or RNA-seq.

organism: Drosophila melanogaster

growth_protocol: ChIP-seq & ATAC-seq: D. melanogaster larvae were grown at 25 degree Celsius on molasses food for 5-6 days.

extract_protocol:
SELEX-seq: All samples were assembled at RT for 30 mins. Procedures followed as described previously (Slattery, Cell, 2011; Kribelbauer, Cell Rep, 2017). ChIP-seq: Inverted larvae were cross-linked at room temperature (RT) for 10 min in 10 ml 1% formaldehyde solution buffered with 50mM HEPES (pH=8.0). Samples were quenched with 1 ml 2.5M Glycine and washed for 5 minutes in quench-solution (125 mM glycine, in 1X PBS and 0.01% Triton X-100). Inverted and cross-linked larvae were washed twice with Buffer A (10mM HEPES, pH=8.0; 10mM EDTA, pH=8.0, 0.5mM EGTA, pH=8.0; 0.025 % Triton-X) and twice with Buffer B (10mM HEPES, pH=8.0; 200mM NaCl, 1mM EDTA, pH=8.0; 0.5mM EGTA, 0.01 % Triton X-100). Wing discs were detached on ice in Buffer B and transferred into a final volume of 1 ml Buffer C (10mM HEPES, pH=8.0 ;1mM EDTA, pH=8.0; 0.5mM EGTA, pH=8.0). Chromatin was sheared into fragments by using a probe sonicator at 15 % amplitude (total time: 12 min with 15 seconds on and 40 second off intervals) and flash-frozen in liquid nitrogen for storage at -80℃ until further processing. Sheared chromatin was diluted in 5X RIPA dilution buffer (1x RIPA: 140mM NaCl; 10mM HEPES, pH=8.0; 1mM EDTA, pH=8.0; 1 % Glycerol; 1% Triton X-100; 0.1% DOC) and blocked with 10μg of the respective IgG-coated magnetic beads (Dynabeads, ThermoFisher) for 1h at 4℃. Beads were removed with a magnetic stand and supernatant was transferred into a new tube. 10 % of the sample was set aside to serve as an input control. Specific antibody and 1 % of Bovine Serum Albumine (BSA) was added to the remaining chromatin and incubated over night at 4℃. 30 μg of IgG-coated and pre-blocked (with 1 % BSA) Dynabeads were added to each chromatin antibody solution and incubated for another 2 hours. Antibody-bound TF-chromatin complexes were isolated by magnetic separation (5 min on a magnetic stand) and beads were washed twice with 1x RIPA, once with high salt RIPA (500mM NaCl), once with LiCl-Buffer and once with TE (10 mM Tris-Base, pH=8.0; 1mM EDTA, pH=8.0). Beads with chromatin and the input sample were redissolved in 0.5 ml Elution-Buffer (TE with 0.5 % Sodium Dodecyl Sulfate (SDS) and 50mM NaCl) and incubated for 30 min at 37℃ with RNase, followed by 2 hours at 55℃ with proteinase K (ThermoFisher). Remaining DNA-protein complexes were decrosslinked by incubating for 16 hours at 65℃. DNA was separated from the Dynabeads by magnetic separation and purified by phenol:chloroform extraction and DNA precipitation using 1x volume of isopropanol in 100mM ammonium acetate and 1 μl glycogen. Precipitated DNA was redissolved in 30 μl TE. ATAC-seq: Wing imaginal discs of third instar larvae were dissected from a lab stock of yw genotype in Phosphate-Buffered-Saline. Discs were washed in nuclear extraction buffer (NEB, 10nM HEPES pH. 7.5, 2.5mM MgCL2, 10mM KCl) and placed in a 1mL dounce homogenizer (Wheaton) on ice. Discs were treated with 15 strokes of the loose pestle, followed by a 10 minute incubation on ice, then 20 strokes of the tight pestle. Nuclei were counted using a hemocytometer, and 50,000 nuclei were transferred to a fresh Eppendorf containing 1mL of NEB buffer +0.1% tween-20. Following a brief mixing the nuclei were immediately pelleted for 10 min at a speed of 1000xg. The pellet was re-suspended in ATAC transposition buffer as in (Buenrostro et al., 2015) and tagmentation was carried out as previously described (Buenrostro et al., 2015). Amplified libraries were purified, and size-selected using  ampureXP (Beckman) beads for  a double-sided selection.
Extrated SELEX-seq libraries were amplified using Illumina compatible amplification primers (Illumina small RNAseq or NEBNext Mulitplex Oligos). ChIP-seq and ATAC-seq libraries were constructed using the NEBNext Ultra DNA Library Prep Kit for Illumina with NEBNext Mulitplex Oligos (one separate index per sample) following standard instructions. Libraries were sequenced either at the NY Genome Center (HiSeq-2000) or at Columbia University (NextSeq 500, 75 bp kit).

value: accessible chromatin


title: GS293_RPM


title: IP_Hth_ChIP_wing

value: Hth

tag: antibody

value: anti-Hth (gp52, N-terminal)


title: INPUT_Hth_ChIP_wing

value: input


title: INPUT_Antp_GFP_ChIP_wing

value: Antp-GFP


title: BS19_CD11bhi_YFP-

tag: strain

value: kidney

value: CD11bhiF4/80low YFP-

value: CD45+ Ly6C- MHCII+ CD64+ F4/80low CD11bhi YFP-


title: IP_Exd_V5_wt_rep2_ChIP_wing

value: Exd-V5 Transgene

value: anti-V5 (Invitrogen)


title: IP_Antp_GFP_ChIP_wing

value: anti-GFP (Invitrogen)


title: INPUT_Exd_R2AR5A_V5_rep1_ChIP_wing

value: Exd-R2AR5A-V5 Transgene


title: IP_Exd_R2AR5A_V5_rep2_ChIP_wing


title: INPUT_Exd_V5_wt_rep2_ChIP_wing


title: INPUT_Exd_V5_wt_rep1_ChIP_wing


title: IP_Exd_R2AR5A_V5_rep1_ChIP_wing


title: INPUT_Exd_R2AR5A_V5_rep2_ChIP_wing


title: RPE, Macula AMD SAM533

status: Public on Jan 22 2020

city: South San Francisco

first: Luz

middle: D

last: Orozco

address: 1 DNA Way

department: Bioinformatics

institute: Genentech Inc.

data_processing: Reads were aligned to the GRCh38 genome using GSNAP and genes were counted using the HTSeqGenie Bioconductor package. Gene models were from our internal database and available in the manuscript supplementary material. "nRPKM" normalization was performed. This is similar to "RPKM" normalization but includes an extra step for sizeFactor adjustment and is described in the Methods of the manuscript. Supplementary files use Ensembl IDs, counts and nRPKM statistics.

source_name: RNA from the cell pellet

extract_protocol: Cells were centrifuged at 2,600 rcf for 7 minutes (neurons) or 6,600 rcf for 7 minutes (other cell types) and the supernatant was discarded. RNA was extracted from the cell pellet using Qiagen RNeasy Plus Micro kit in a final volume of 25 μl. We typically obtained 200-300 ng RNA from neurons, 10-20 ng from microglia, and 2-5 ng from astrocytes. RNA quality was quantified using the Bioanalyzer.

value: RPE, Macula

value: AMD

tag: samid


title: Retina, non-Macula Control SAM537

tag: tissue

value: Retina, non-Macula

value: Control


title: RPE, non-Macula Control SAM536

value: RPE, non-Macula


title: RPE, non-Macula Control SAM535


title: IP_Exd_V5_wt_rep1_ChIP_wing

tag: genotype/variation


title: Retina, non-Macula Control SAM534


title: Retina, non-Macula AMD SAM531


title: RPE, Macula Control SAM530


title: RPE, non-Macula Control SAM529


title: RPE, Macula AMD SAM528


title: RPE, Macula Control SAM527


title: Retina, Macula Control SAM526

value: Retina, Macula


title: Retina, Macula Control SAM525


title: Retina, non-Macula AMD SAM524


title: Retina, non-Macula Control SAM523


title: Retina, non-Macula Control SAM521


title: RPE, non-Macula Control SAM522


title: RPE, Macula Control SAM519


title: Retina, non-Macula Control SAM520


title: Retina, Macula AMD SAM517


title: Retina, non-Macula Control SAM516


title: Retina, non-Macula Control SAM514


title: RPE, non-Macula Control SAM515


title: RPE, non-Macula Control SAM513


title: RPE, Macula Control SAM512


title: RPE, non-Macula Control SAM508


title: RPE, non-Macula Control SAM511


title: Retina, Macula Control SAM532


title: Retina, Macula Control SAM510


title: Retina, Macula Control SAM504


title: RPE, non-Macula Control SAM503


title: RPE, non-Macula AMD SAM505


title: Retina, Macula Control SAM507


title: Retina, Macula Control SAM509


title: Retina, non-Macula Control SAM501


title: RPE, Macula Control SAM502


title: Retina, Macula Control SAM500


title: Retina, Macula Control SAM498


title: Retina, non-Macula AMD SAM499


title: Retina, Macula Control SAM506


title: Retina, Macula Control SAM497


title: Retina, non-Macula Control SAM496


title: RPE, Macula AMD SAM495


title: RPE, Macula Control SAM494


title: RPE, Macula Control SAM493


title: RPE, Macula Control SAM518


title: RPE, non-Macula Control SAM490


title: RPE, non-Macula Control SAM491


title: Retina, Macula Control SAM487


title: Retina, non-Macula Control SAM486


title: RPE, non-Macula Control SAM488


title: Retina, Macula Control SAM485


title: RPE, Macula Control SAM489


title: Retina, Macula Control SAM484


title: Retina, non-Macula Control SAM483


title: RPE, non-Macula Control SAM481


title: RPE, non-Macula AMD SAM478


title: RPE, Macula Control SAM477


title: Retina, Macula AMD SAM476


title: RPE, non-Macula Control SAM474


title: Retina, Macula Control SAM480


title: RPE, non-Macula Control SAM473


title: RPE, non-Macula Control SAM471


title: Retina, Macula Control SAM470


title: RPE, Macula Control SAM492


title: Retina, Macula Control SAM469


title: Retina, non-Macula Control SAM472


title: Retina, Macula AMD SAM466


title: RPE, non-Macula Control SAM465


title: RPE, non-Macula Control SAM464


title: Retina, Macula Control SAM463


title: RPE, Macula Control SAM462


title: Retina, non-Macula Control SAM461


title: RPE, Macula Control SAM457


title: RPE, non-Macula Control SAM458


title: RPE, Macula AMD SAM479


title: Retina, Macula AMD SAM456


title: RPE, Macula AMD SAM454


title: Retina, non-Macula Control SAM455


title: RPE, non-Macula AMD SAM453


title: Retina, non-Macula Control SAM452


title: Retina, non-Macula Control SAM475


title: RPE, non-Macula Control SAM450


title: Retina, Macula Control SAM451


title: Retina, Macula Control SAM449


title: Retina, non-Macula Control SAM468


title: Retina, non-Macula Control SAM467


title: RPE, Macula Control SAM442


title: Retina, non-Macula Control SAM443


title: RPE, Macula Control SAM446


title: RPE, Macula Control SAM447


title: RPE, Macula Control SAM444


title: RPE, Macula Control SAM448


title: Retina, non-Macula Control SAM439


title: Retina, non-Macula Control SAM440


title: RPE, Macula Control SAM441


title: RPE, non-Macula Control SAM438


title: RPE, non-Macula Control SAM460


title: RPE, non-Macula Control SAM459


title: RPE, Macula AMD SAM437


title: Retina, non-Macula Control SAM436


title: Retina, non-Macula Control SAM435


title: Retina, non-Macula Control SAM434


title: Retina, non-Macula AMD SAM432


title: RPE, non-Macula Control SAM433


title: Retina, non-Macula AMD SAM430


title: Retina, non-Macula Control SAM445


title: RPE, Macula Control SAM427


title: RPE, Macula Control SAM429


title: Retina, Macula Control SAM426


title: RPE, non-Macula Control SAM428


title: Retina, non-Macula Control SAM482


title: Retina, Macula Control SAM422


title: Retina, Macula Control SAM423


title: RPE, non-Macula Control SAM420


title: Retina, Macula AMD SAM421


title: RPE, non-Macula Control SAM417


title: Retina, Macula Control SAM412


title: RPE, Macula Control SAM413


title: RPE, Macula Control SAM414


title: RPE, Macula Control SAM411


title: Retina, Macula AMD SAM409


title: RPE, Macula Control SAM419


title: RPE, Macula Control SAM408


title: Retina, Macula Control SAM415


title: RPE, non-Macula Control SAM406


title: Retina, Macula Control SAM407


title: RPE, non-Macula Control SAM418


title: Retina, non-Macula Control SAM425


title: Retina, Macula Control SAM404


title: RPE, non-Macula AMD SAM405


title: Retina, Macula Control SAM431


title: RPE, Macula Control SAM403


title: Retina, non-Macula AMD SAM402


title: Retina, Macula Control SAM401


title: Retina, Macula Control SAM399


title: Retina, Macula Control SAM424


title: RPE, Macula Control SAM400


title: RPE, Macula AMD SAM398


title: Retina, Macula Control SAM416


title: Retina, non-Macula AMD SAM396


title: RPE, non-Macula Control SAM397


title: Retina, Macula AMD SAM395


title: Retina, Macula Control SAM394


title: Retina, non-Macula Control SAM389


title: Retina, non-Macula Control SAM390


title: Retina, Macula Control SAM388


title: RPE, non-Macula Control SAM387


title: Retina, non-Macula Control SAM391


title: Retina, Macula AMD SAM392


title: Retina, non-Macula AMD SAM382


title: RPE, Macula Control SAM380


title: Retina, non-Macula Control SAM379


title: Retina, Macula Control SAM386


title: RPE, Macula AMD SAM378


title: RPE, non-Macula Control SAM385


title: Retina, Macula Control SAM384


title: RPE, non-Macula Control SAM372


title: Retina, Macula Control SAM375


title: RPE, Macula Control SAM381


title: Retina, Macula Control SAM370


title: Retina, non-Macula Control SAM371


title: RPE, non-Macula AMD SAM377


title: RPE, Macula Control SAM369


title: RPE, Macula Control SAM368


title: Retina, non-Macula Control SAM374


title: Retina, Macula Control SAM373


title: Retina, Macula AMD SAM410


title: Retina, non-Macula Control SAM383


title: RPE, Macula AMD SAM366


title: RPE, non-Macula Control SAM361


title: RPE, non-Macula Control SAM362


title: RPE, non-Macula Control SAM359


title: RPE, non-Macula Control SAM358


title: Retina, non-Macula AMD SAM360


title: RPE, Macula Control SAM376


title: RPE, non-Macula Control SAM393


title: Retina, non-Macula Control SAM364


title: Retina, non-Macula Control SAM365


title: Retina, Macula Control SAM355


title: RPE, non-Macula Control SAM353


title: RPE, non-Macula Control SAM354


title: RPE, non-Macula Control SAM351


title: RPE, Macula Control SAM357


title: RPE, non-Macula Control SAM348


title: RPE, Macula Control SAM346


title: RPE, non-Macula Control SAM352


title: RPE, Macula Control SAM347


title: Retina, Macula Control SAM344


title: Retina, non-Macula Control SAM367


title: Retina, non-Macula Control SAM343


title: Retina, Macula Control SAM345


title: Retina, Macula Control SAM363


title: RPE, Macula Control SAM356


title: Retina, non-Macula Control SAM349


title: Retina, non-Macula Control SAM350


title: RPE, Macula Control SAM341


title: RPE, non-Macula Control SAM340


title: RPE, Macula Control SAM338


title: RPE, Macula AMD SAM337


title: Retina, non-Macula Control SAM339


title: Retina, non-Macula Control SAM336


title: Retina, Macula Control SAM334


title: Retina, non-Macula Control SAM333


title: Retina, non-Macula Control SAM332


title: Retina, non-Macula Control SAM331


title: RPE, non-Macula Control SAM326


title: Retina, Macula Control SAM317


title: RPE, Macula Control SAM316


title: RPE, Macula Control SAM318


title: RPE, non-Macula Control SAM312


title: RPE, Macula AMD SAM315


title: Retina, Macula AMD SAM322


title: Retina, Macula Control SAM327


title: RPE, non-Macula Control SAM319


title: Retina, Macula Control SAM311


title: Retina, non-Macula AMD SAM321


title: Retina, non-Macula Control SAM309


title: Retina, Macula Control SAM328


title: RPE, non-Macula Control SAM307


title: Retina, Macula Control SAM306


title: Retina, non-Macula Control SAM308


title: RPE, non-Macula Control SAM304


title: RPE, Macula Control SAM320


title: Retina, Macula Control SAM324


title: Retina, non-Macula Control SAM323


title: RPE, Macula Control SAM335


title: Retina, non-Macula Control SAM342


title: Retina, Macula Control SAM310


title: Retina, non-Macula Control SAM325


title: RPE, Macula Control SAM330


title: RPE, non-Macula Control SAM305


title: RPE, Macula Control SAM329


title: Retina, non-Macula Control SAM303


title: RPE, Macula Control SAM314


title: RPE, non-Macula Control SAM313


title: Retina, non-Macula Control SAM302


title: Retina, non-Macula Control SAM299


title: RPE, Macula AMD SAM300


title: RPE, Macula Control SAM301


title: RPE, Macula Control SAM293


title: Retina, non-Macula Control SAM297


title: Retina, Macula Control SAM295


title: Retina, Macula AMD SAM291


title: RPE, non-Macula Control SAM298


title: RPE, non-Macula Control SAM288


title: RPE, Macula Control SAM294


title: RPE, non-Macula Control SAM292


title: RPE, non-Macula Control SAM296


title: RPE, Macula Control SAM286


title: Retina, non-Macula Control SAM289


title: Retina, Macula Control SAM281


title: RPE, non-Macula Control SAM279


title: RPE, non-Macula Control SAM285


title: RPE, Macula AMD SAM282


title: RPE, Macula Control SAM287


title: RPE, non-Macula Control SAM283


title: RPE, Macula Control SAM277


title: Retina, non-Macula Control SAM278


title: Retina, Macula Control SAM276


title: Retina, Macula Control SAM273


title: RPE, non-Macula Control SAM274


title: Retina, Macula Control SAM290


title: Retina, Macula Control SAM272


title: Retina, non-Macula Control SAM270


title: Retina, Macula Control SAM269


title: Retina, non-Macula Control SAM271


title: RPE, non-Macula Control SAM267


title: RPE, non-Macula Control SAM259


title: RPE, Macula Control SAM268


title: RPE, Macula Control SAM280


title: RPE, non-Macula Control SAM265


title: RPE, non-Macula Control SAM261


title: Retina, Macula Control SAM263


title: Retina, non-Macula AMD SAM262


title: Retina, Macula Control SAM266


title: RPE, Macula Control SAM256


title: Retina, non-Macula Control SAM258


title: RPE, Macula Control SAM254


title: Retina, Macula Control SAM284


title: RPE, Macula AMD SAM251


title: RPE, non-Macula Control SAM249


title: Retina, Macula AMD SAM252


title: RPE, Macula Control SAM257


title: RPE, Macula Control SAM250


title: Retina, non-Macula Control SAM253


title: Retina, Macula Control SAM255


title: Retina, non-Macula Control SAM275


title: RPE, non-Macula AMD SAM264


title: RPE, Macula Control SAM248


title: Retina, Macula Control SAM247


title: Retina, Macula Control SAM246


title: Retina, non-Macula Control SAM245


title: RPE, non-Macula AMD SAM260


title: Retina, Macula Control SAM244


title: RPE, non-Macula AMD SAM243


title: RPE, non-Macula Control SAM242


title: RPE, non-Macula Control SAM240


title: Retina, non-Macula Control SAM241


title: Retina, Macula Control SAM238


title: Retina, Macula Control SAM236


title: RPE, non-Macula Control SAM235


title: Retina, non-Macula Control SAM237


title: Retina, non-Macula Control SAM234


title: RPE, non-Macula Control SAM239


title: Retina, non-Macula Control SAM232


title: RPE, non-Macula Control SAM231


title: RPE, Macula Control SAM230


title: RPE, Macula Control SAM227


title: Retina, non-Macula Control SAM224


title: RPE, non-Macula AMD SAM228


title: RPE, non-Macula Control SAM226


title: RPE, non-Macula Control SAM222


title: Retina, non-Macula Control SAM223


title: Retina, Macula Control SAM229


title: RPE, non-Macula Control SAM220


title: RPE, non-Macula Control SAM225


title: Retina, Macula Control SAM218


title: RPE, Macula Control SAM221


title: Retina, non-Macula Control SAM219


title: Retina, non-Macula Control SAM217


title: RPE, non-Macula AMD SAM216


title: Retina, non-Macula AMD SAM215


title: Retina, Macula Control SAM213


title: RPE, Macula Control SAM214


title: Retina, Macula Control SAM212


title: Retina, non-Macula Control SAM233


title: RPE, non-Macula Control SAM211


title: RPE, non-Macula Control SAM206


title: RPE, Macula Control SAM210


title: Retina, non-Macula Control SAM209


title: RPE, non-Macula Control SAM205


title: Retina, Macula Control SAM204


title: RPE, Macula Control SAM203


title: RPE, non-Macula Control SAM202


title: RPE, non-Macula AMD SAM199


title: RPE, non-Macula Control SAM201


title: Retina, Macula Control SAM196


title: RPE, Macula Control SAM198


title: Retina, non-Macula Control SAM195


title: RPE, non-Macula Control SAM197


title: Retina, non-Macula AMD SAM200


title: RPE, non-Macula Control SAM194


title: RPE, non-Macula Control SAM192


title: Retina, non-Macula AMD SAM189


title: Retina, non-Macula Control SAM193


title: RPE, non-Macula Control SAM191


title: Retina, Macula AMD SAM188


title: Retina, non-Macula Control SAM190


title: RPE, Macula Control SAM186


title: RPE, Macula Control SAM187


title: Retina, non-Macula AMD SAM184


title: Retina, Macula Control SAM185


title: Retina, Macula Control SAM207


title: RPE, Macula Control SAM182


title: Retina, non-Macula Control SAM208


title: RPE, non-Macula Control SAM183


title: RPE, Macula Control SAM181


title: RPE, Macula Control SAM179


title: RPE, non-Macula Control SAM180


title: RPE, Macula Control SAM178


title: Retina, Macula Control SAM177


title: RPE, Macula Control SAM176


title: Retina, Macula Control SAM175


title: RPE, Macula Control SAM172


title: Retina, non-Macula Control SAM171


title: RPE, Macula Control SAM173


title: Retina, non-Macula Control SAM167


title: Retina, non-Macula AMD SAM168


title: RPE, Macula Control SAM166


title: RPE, Macula Control SAM169


title: Retina, Macula Control SAM163


title: RPE, Macula Control SAM165


title: RPE, Macula Control SAM161


title: Retina, Macula Control SAM160


title: RPE, Macula Control SAM158


title: Retina, Macula Control SAM170


title: Retina, Macula Control SAM159


title: RPE, non-Macula Control SAM156


title: RPE, Macula Control SAM157


title: Retina, Macula Control SAM162


title: Retina, non-Macula Control SAM155


title: Retina, non-Macula Control SAM153


title: Retina, non-Macula Control SAM154


title: Retina, non-Macula Control SAM151


title: RPE, non-Macula Control SAM148


title: Retina, non-Macula Control SAM147


title: Retina, Macula Control SAM174


title: Retina, Macula Control SAM146


title: Retina, non-Macula Control SAM145


title: RPE, Macula Control SAM144


title: RPE, Macula Control SAM142


title: RPE, Macula Control SAM140


title: Retina, non-Macula Control SAM141


title: RPE, non-Macula Control SAM139


title: Retina, non-Macula Control SAM137


title: RPE, Macula Control SAM136


title: Retina, non-Macula Control SAM133


title: Retina, Macula AMD SAM135


title: Retina, Macula Control SAM134


title: Retina, Macula AMD SAM132


title: RPE, non-Macula Control SAM152


title: RPE, non-Macula Control SAM129


title: Retina, Macula Control SAM131


title: Retina, non-Macula Control SAM130


title: RPE, Macula Control SAM150


title: Retina, non-Macula Control SAM128


title: Retina, non-Macula Control SAM127


title: RPE, non-Macula Control SAM125


title: RPE, non-Macula Control SAM149


title: Retina, Macula Control SAM126


title: RPE, non-Macula Control SAM123


title: RPE, non-Macula AMD SAM124


title: Retina, Macula AMD SAM122


title: Retina, non-Macula AMD SAM120


title: Retina, Macula Control SAM143


title: Retina, non-Macula Control SAM119


title: Retina, non-Macula Control SAM138


title: Retina, Macula AMD SAM118


title: Retina, Macula Control SAM117


title: RPE, Macula Control SAM115


title: RPE, Macula AMD SAM116


title: Retina, non-Macula AMD SAM121


title: Retina, non-Macula AMD SAM113


title: RPE, Macula Control SAM112


title: Retina, Macula AMD SAM110


title: RPE, Macula AMD SAM109


title: RPE, non-Macula AMD SAM108


title: RPE, Macula AMD SAM106


title: Retina, non-Macula Control SAM103


title: RPE, Macula AMD SAM107


title: Retina, non-Macula AMD SAM105


title: RPE, Macula Control SAM101


title: RPE, non-Macula AMD SAM100


title: Retina, Macula AMD SAM102


title: RPE, non-Macula AMD SAM99


title: RPE, Macula AMD SAM98


title: RPE, Macula AMD SAM97


title: RPE, non-Macula AMD SAM95


title: RPE, non-Macula Control SAM96


title: RPE, non-Macula AMD SAM94


title: Retina, Macula AMD SAM93


title: RPE, non-Macula Control SAM90


title: RPE, non-Macula AMD SAM114


title: RPE, Macula AMD SAM91


title: Retina, non-Macula Control SAM89


title: RPE, non-Macula AMD SAM88


title: Retina, non-Macula AMD SAM86


title: Retina, non-Macula Control SAM111


title: Retina, non-Macula Control SAM84


title: Retina, Macula Control SAM104


title: Retina, Macula AMD SAM83


title: RPE, Macula Control SAM79


title: Retina, Macula AMD SAM82


title: RPE, non-Macula Control SAM81


title: RPE, Macula Control SAM80


title: Retina, Macula Control SAM77


title: Retina, Macula Control SAM76


title: Retina, non-Macula Control SAM164


title: Retina, Macula AMD SAM78


title: RPE, non-Macula Control SAM75


title: Retina, Macula AMD SAM73


title: Retina, non-Macula AMD SAM92


title: Retina, Macula Control SAM74


title: Retina, non-Macula Control SAM87


title: RPE, Macula AMD SAM71


title: RPE, non-Macula AMD SAM69


title: RPE, non-Macula Control SAM85


title: RPE, Macula AMD SAM70


title: Retina, Macula AMD SAM68


title: Retina, non-Macula AMD SAM66


title: RPE, Macula AMD SAM67


title: RPE, non-Macula Control SAM64


title: Retina, non-Macula AMD SAM63


title: Retina, Macula Control SAM72


title: RPE, non-Macula AMD SAM62


title: Retina, Macula AMD SAM60


title: RPE, Macula Control SAM61


title: Retina, non-Macula AMD SAM59


title: Retina, non-Macula AMD SAM58


title: Muscle_CB1KO_HFHS_replicate_4

hyb_protocol: A total of 600 ng Cy3-labeled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 microL containing 0.5x Agilent fragmentation buffer and 2x Agilent gene expression blocking agent following the manufacturers instructions.  On completion of the fragmentation reaction, 25 microL of 2× Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint GE3 Mouse 8x60k v1 gene expression microarrays (G4852-60510) for 17 hours at 65°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), and dried by slowly removing from Wash buffer 2.

scan_protocol: Following posthybridization rinses, arrays were scanned using an Agilent SureScan microarray Scanner at 5 micron, and hybridization intensity data extracted from the scanned images using Agilent’s Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.

data_processing: Raw microarray hybridization intensity data from four separate experiments were log-transformed.  Raw microarray data were log transformed to yield z-scores. The z-ratio was calculated as the difference between the observed gene z-scores for the experimental and the control comparisons, and dividing by the standard deviation associated with the distribution of these differences. Z-ratio values of ± 1.5 were used as cut-off values and calculated using a 5% false discovery rate (FDR) threshold. The complete set was tested for gene set enrichment using parametric analysis of gene set enrichment. For each pathway z-score, a p-value was computed using JMP 6.0 software to test for the statistical significance.

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng total RNA using the Low-Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were evaluated with the NanoDrop ND-1000 Spectrophotometer (Wilmington, DE).

growth_protocol: Skeletal muscle-specific CB1R knockout (Skm-CB1R-/-) and control littermates (Skm-CB1R+/+) mice were developed by crossing a LoxP-flanked CB1R gene (CNR1) mouse with an ACTA-Cre (Cre under control of ACTA1 promoter). Ablation of CB1R in skeletal muscle but not CNR1flox/flox mice were bred as described previously. Mice expressing Cre under the ACTA1 gene promoter (Acta-Cre) were obtained from Jackson Laboratories (Bar Harbor, ME). Acta-Cre and CNR1flox/flox mice were bred to obtain CNR1flox/flox-Cre+ (Skm-CB1R-/-) mice and CNR1flox/flox-Cre+ (Skm-CB1R+/+) control littermates. Mice were housed in groups of 4 using 12 h dark/light cycles, provided with water and fed ad libitum.

extract_protocol: Total RNA was isolated from flash-frozen, dissected skeletal muscles tissue using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturers protocol. Total RNA quantity and quality was tested using the Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA).

treatment_protocol: Age and sex matched littermate mice were randomly assigned to vehicle or S961 and to SD or HFHS groups, and fed either a standard diet (SD; 16.7% kJ fat and 12.4% kJ sugar wt/wt) or a high fat-high sugar diet (HFHS; 49.2% kJ fat and 32.2% kJ sugar wt/wt) (Dyets Inc, Bethlehem, PA) to induce obesity. Body weight and food intake were measured weekly in the diet studies. Tissues were collected after 15 weeks of diet.

value: male

value: 21-23 weeks

value: CB1 KO; Skm-CB1R-/-

tag: diet

value: High fat-high sugar diet

value: Muscle


title: Muscle_WT_HFHS_replicate_4

value: Wildtype; Skm-CB1R+/+

value: Standard diet


title: RPE, Macula Control SAM57

tag: age


title: Muscle_WT_HFHS_replicate_2


title: Muscle_CB1KO_HFHS_replicate_2


title: Muscle_WT_HFHS_replicate_3


title: Retina, Macula Control SAM55


title: Muscle_CB1KO_HFHS_replicate_1


title: Muscle_CB1KO_SD_replicate_4


title: Muscle_WT_HFHS_replicate_1


title: Muscle_CB1KO_SD_replicate_2


title: Muscle_WT_SD_replicate_4


title: Muscle_CB1KO_SD_replicate_3


title: Muscle_WT_SD_replicate_3


title: Retina, non-Macula AMD SAM65


title: Muscle_CB1KO_HFHS_replicate_3


title: RPE, Macula AMD SAM56


title: Retina, Macula AMD SAM54


title: Retina, non-Macula AMD SAM51


title: RPE, non-Macula AMD SAM50


title: RPE, Macula AMD SAM52


title: Retina, non-Macula Control SAM49


title: RPE, non-Macula AMD SAM48


title: Muscle_WT_SD_replicate_1


title: Retina, non-Macula Control SAM40


title: RPE, Macula Control SAM42


title: RPE, non-Macula Control SAM41


title: Retina, non-Macula Control SAM43


title: Retina, non-Macula Control SAM39


title: RPE, Macula Control SAM44


title: RPE, non-Macula Control SAM37


title: Retina, Macula Control SAM32


title: Retina, non-Macula Control SAM38


title: RPE, Macula Control SAM31


title: Retina, Macula Control SAM35


title: RPE, Macula Control SAM34


title: RPE, Macula Control SAM33


title: RPE, Macula Control SAM28


title: RPE, non-Macula Control SAM30


title: RPE, non-Macula Control SAM36


title: Retina, non-Macula Control SAM25


title: RPE, non-Macula Control SAM26


title: Retina, non-Macula Control SAM29


title: Retina, Macula Control SAM24


title: RPE, Macula Control SAM46


title: RPE, Macula Control SAM53


title: RPE, non-Macula Control SAM45


title: Retina, Macula Control SAM21


title: RPE, non-Macula Control SAM22


title: RPE, Macula Control SAM20


title: Retina, Macula Control SAM16


title: Retina, non-Macula Control SAM15


title: RPE, non-Macula Control SAM19


title: Retina, non-Macula Control SAM14


title: Muscle_WT_SD_replicate_2


title: RPE, non-Macula Control SAM12


title: Retina, non-Macula Control SAM11


title: RPE, non-Macula Control SAM10


title: Retina, non-Macula Control SAM13


title: Retina, non-Macula Control SAM9


title: Retina, non-Macula Control SAM17


title: Retina, Macula Control SAM7


title: RPE, non-Macula Control SAM23


title: RPE, Macula Control SAM6


title: Muscle_CB1KO_SD_replicate_1


title: Retina, non-Macula Control SAM3


title: RPE, non-Macula Control SAM2


title: RPE, non-Macula Control SAM4


title: Retina, non-Macula Control SAM8


title: RPE, Macula Control SAM1