title: MLL-AF9_ChIPSeq

city: Birmingham

first: Salam

middle: Adli

last: Assi

address: IBR

department: Institute for Cancer and Genomic Sciences

institute: University of Birmingham

description: human CD34+ hematopoietic stem and progenitor cells expressing MLL-Af4

data_processing:
Quality of reads was verified using FASTQC analysis
Sequences reads in fastq format were mapped onto the reference human genome version hg38, Genome Reference Consortium GRCh38. The Illumina reads were aligned to the human genome using Bowtie2
Reads that were uniquely aligned to chromosomal positions were retained and duplicate reads were removed from the aligned data using Picard tools.
The filtered aligned reads were used to generate density profiles using “genomeCoverageBed” function from bedtools and wigToBigWig was used to generate the bigWig file
Genome_build: hg38
Supplementary_files_format_and_content: bigWig

source_name: FLAG MLL-AF9  ChIPseq

growth_protocol: Human CD34+ hematopoietic stem and progenitor cells expressing MLL-AF9 were transplanted into NSG mice, the CD19+ leukemic cells were extracted from mouse bone marrow for ChIP-seq

extract_protocol:
ChIP-Seq assay were performed essentially as previously described (Ptasinska et al., 2014). Antibody against Flag (Sigma, M2)  was used for IP. Cells were first washed with PBS and then cross-linked with 0.83 mg/ml Di(N-succinimidyl) glutarate (DSG) (Sigma) for 45 minutes at room temperature with rotation. Cells were washed four times with PBS and further crosslinked in PBS with 1% formaldehyde (~0.34 M) for 10 minutes at room temperature. All crosslinking reactions were quenched by adding 4 volumes of PBS and 0.125 M glycine, cells were washed twice in PBS and incubated with Buffer A (10 mM HEPES pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, proteinase inhibitor cocktail (Roche UK, Burgess Hill, UK) and 0.1 mM PMSF), incubated for 10 min at 4°C with rotation, and centrifuged 5 min at 500 x g at 4 °C. The pellet was resuspended in 10 ml of ice–cold Buffer B (10 mM HEPES pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton X-100, protease inhibitor cocktail and 0.1 mM PMSF), incubated for 10 min at 4 °C with rotation and centrifuged for 5 min at 500 x g at 4 °C. Cells were resuspended in 600 μl of ice-cold ChIP lysis buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.25% SDS, protease inhibitor cocktail and 0.1 mM PMSF), incubated 10 min on ice and sonicated at 5 °C using a Bioruptor™ (Diagenode) to generate fragments an average length of 500 bp (10 min with 30 s “ON” and “OFF” cycles, power setting high). The lysates were centrifuged for 5 min at 16,000 x g at 4 °C and the supernatants were diluted with two volumes of ice-cold ChIP dilution buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 7.5% glycerol, protease inhibitor cocktail and 0.1 mM PMSF). For each IP, 15 μl of Dynabeads® protein G (Dynal) were pre–incubated with 50 μg BSA and 1-2 μg antibody for 2 h at 4 °C with rotation. The blocked antibody-bound protein G mix was added to 20–25 μg chromatin in a total volume of 500 μl diluted ChIP lysis buffer and incubated for 2 h at 4°C with rotation. After magnetic separation the beads were washed once with 1 ml wash buffer 1 (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), twice with 1 ml wash buffer 2 (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with 1 ml LiCl buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na-deoxycholate) and twice with 1 ml TE/NaCl buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA). For each wash the beads were mixed with ice-cold washing buffers for 10 min at 4 °C. The immunoprecipitated DNA was eluted two times with 50 μl ChIP elution buffer (100 mM NaHCO3, 1% SDS) for 15 min at RT with shaking. At this step the input control (1% of the starting material) was included in the experimental procedure after first adjusting the final volume to 100 μl with ChIP elution buffer. The eluted DNA was incubated overnight at 65 °C in the presence of 50 μg proteinase K. The DNA was finally purified using Agencourt AMPure (Beckman Coulter) magnetic beads according to the manufacturer’s instructions, eluted with 50 μl x TE and analysed by qPCR using SYBR Green reagents.
KAPA Hyper Prep Kit, Kapa biosystems

treatment_protocol: untreated cells

value: human CD34+

value: control untreated

value: Flag


title: M407 DMSO

status: Public on Apr 30 2018

type: SRA

city: Los Angeles

first: Jennifer

last: Tsoi

state: CA

address: 570 Westwood Blvd, CNSI 4338

department: University of California, Los Angeles

country: USA

institute: UCLA

library_source: transcriptomic

data_processing:
Paired end 50 bp reads generated from the melanoma cell line RNA sequencing were mapped using HISAT2 to the Homo sapiens hg38 genome build and raw counts per quantified using HTSeq.
Raw counts were normalized to FPKM values using conditional quantile normalization (CQN) to adjust for gene length and GC content.
genome build: hg38
processed data files format and content: Matrix of gene expression measurements in FPKM

molecule: total RNA

organism: Homo sapiens

source_name: Melanoma cell line

extract_protocol:
AllPrep DNA/RNA Mini kit from Qiagen
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cells have been treated with DMSO for 48 hours.

tag: cell line

tag: disease state

value: Melanoma


title: B-53

city: East Lansing

first: Timothy

last: Zacharewski

state: Michigan

address: 603 Wilson Road

department: BMB

institute: Michigan State University

description: Mice dosed every 4 days for 28 days

data_processing:
Quality control: FASTQC v0.11.3
Removal of artifacts: FASTX v0.0.14
Alignment: TopHat2 v2.1.0
Conversion to SAM format: SAMTools v0.1.19
Abundance measurement: HTSeq v0.6.1
Count transformation: DESeq
Normalization and analysis: Transformed counts were normalized using a semi-parametric approach (Eckel et al., 2004) and analyzed using an empirical Bayes method (Eckel et al., 2005).
Genome_build: GRCm38 release 81
Supplementary_files_format_and_content: Tab-delimited text files represent the number of aligned reads to each gene

organism: Mus musculus

source_name: Femur

extract_protocol:
Frozen femurs were crushed in liquid nitrogen with a Bessman Tissue Pulverizer (Spectrum Laboratories, Rancho Dominguez, CA). Total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. RNA was quantified using a Nano-drop spectrophotometer (Thermo Scientific, Wilmington, DE) at 260 nm. Purity was assessed using the A260/A280 ratio and quality was analyzed using the Caliper LabChip GX (Perkin Elmer, Waltham, MA).
Libraries were were prepared using the Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA) Library sizes were confirmed using Caliper GX and quantified by the Kapa biosystems qPCR according to manufacturers instructions

treatment_protocol: Male C57BL/6 mice were gavaged with sesame oil vehicle, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 ug/kg TCDD every  4 days for 28 days.

tag: strain

tag: Sex

value: Male

tag: age

value: Post natal day 56

tag: tissue

value: Femur

tag: duration of treatment

value: 28 Days

tag: treatment

value: TCDD

tag: dose

tag: dose unit

value: µg/kg


title: B-71

description: Mice dosed every 4 days for 28 days


title: B-61

description: Mice dosed every 4 days for 28 days


title: Kasumi1_RNAseq_2_Erplus

country: United Kingdom

data_processing:
Quality of reads was verified using FASTQC analysis
RNA-Seq reads were aligned to the hg38 human genome build using STAR.
Separate density files for the positive and negative strand were generated for RNA-seq data and uploaded to the UCSC genome browser
Fragments per Kilobase of transcript per Million mapped reads (FPKM) values for each gene were extracted using Cufflinks
Genome_build: hg38
Supplementary_files_format_and_content: bedGraph

source_name: inducible version of CEBPA (C/EBPα-ER) in Kasumi-1 cells treated with E2 (estradiol) replicate 2

growth_protocol: C/EBPα-ER vector required RectroNectin (recombinant human fibronectin fragment, Takara, Japan) assisted transduction. Non-tissue culture treated 6 well plate was incubated with 2μg/ml RetroNectin solution in PBS at 24μg/ml for 2 hours at room temperature. The RectroNectin solution was subsequently removed and the plate blocked ith 2ml PBS with 2% BSA for 20minutes at room temperature. Finally, the PBS/BSA solution was removed and the wells washed with 2ml Hank Balanced Salt solution containing 2.5% HEPES. Concentrated virus was coated onto the wells by centrifugation for 45 minutes at 2000xg at room temperature. Virus loading was repeated once more before 1x106 cells, in media mixed with concentrated virus (50:50 ratio), at a final concentration of 0.5x106 /ml was added to the wells with polybrene at 8μg/ml. The plate was left overnight at 37oC, 5% CO2 in a humidified incubator. On the next day, the cells were centrifuged at 300xg at room temperature for 5 minutes and the cells were re-suspended in standard RPMI 1640 with 10% FCS, supplemented with penicillin, streptomycin and glutamine. CEBPA-ER transduced cells were isolated by FACS using GFP labelling.

extract_protocol:
RNA was isolated from cells by TrizolTM (Life Technologies, US) as by manufacturer’s instructions. At the last step of the protocol RNA was resuspended in 17μl of RNAse free water to which was added 2μl of 10x buffer supplied with the Ambion Turbo DNaseI (Thermos Scientific, USA), of which 1 μl was added. All of which was incubated at 37oC for 30 minutes. The RNA solution was then purified using a Nucleospin RNA clean up column (Machery Nagel, France), according to their instructions. The quality of RNA from all methods was assessed using a spectrophotometer, by the ratio of the absorbance at 260nM and 280nM wavelengths. RNA has a greater absorbance in the 260nM wavelength, Eukaryotic Total RNA PICO Bioanalyser chip (Agilent technologies, USA) allows visualisation of the size of the RNA molecules and thus, demonstrates whether the sample is degraded or not.
RNA-seq libraries were prepared with a Total RNA Ribo-zero library preparation kit (with ribosomal RNA depletion) (Illumina, USA) according to manufacturer’s instructions with the following alterations: 15 cycles of PCR was undertaken to amplify the library and adaptors for multiplexing were used at a 1:4 dilution. Library quality was checked by running the samples on a Bioanalyser and libraries were quantified using a Kapa library quantification kit (Kapa Biosystems, USA) and run in a pool of eight indexed libraries in two lane of a HiSeq 2500 (Illumina, USA) using rapid run chemistry with 100bp paired end reads.

tag: cell type

value: Kasumi-1 cells

tag: genotype

value: inducible version of CEBPA

value: E2 (estradiol)


title: B-72

description: Mice dosed every 4 days for 28 days


title: B-50

description: Mice dosed every 4 days for 28 days


title: B-63

description: Mice dosed every 4 days for 28 days


title: B-67

description: Mice dosed every 4 days for 28 days


title: B-54

description: Mice dosed every 4 days for 28 days


title: Kasumi1_RNAseq_2_Erminus

source_name: inducible version of CEBPA (C/EBPα-ER) in Kasumi1 cells treated with vehicle ethanol replicate 2

value: vehicle ethanol


title: B-41

description: Mice dosed every 4 days for 28 days


title: B-42

description: Mice dosed every 4 days for 28 days


title: B-45

description: Mice dosed every 4 days for 28 days


title: B-64

description: Mice dosed every 4 days for 28 days


title: Kasumi1_RNAseq_2_CTRLplus

source_name: control Kasumi-1 cells treated with E2 (estradiol) replicate 2

value: control Kasumi-1 cells


title: Kasumi1_RNAseq_2_CTRLminus

source_name: control Kasumi-1 cells treated with vehicle ethanol replicate 2


title: B-39

description: Mice dosed every 4 days for 28 days


title: B-37

description: Mice dosed every 4 days for 28 days


title: B-32

description: Mice dosed every 4 days for 28 days


title: Kasumi1_RNAseq_1_CTRLplus

source_name: control Kasumi-1 cells treated with E2 (estradiol) replicate 1


title: B-30

description: Mice dosed every 4 days for 28 days


title: Kasumi1_RNAseq_1_CTRLminus

source_name: control Kasumi-1 cells treated with vehicle ethanol replicate 1


title: B-31

description: Mice dosed every 4 days for 28 days


title: Kasumi1_RNAseq_1_Erminus

source_name: inducible version of CEBPA (C/EBPα-ER) in Kasumi1 cells treated with vehicle ethanol replicate 1


title: B-38

description: Mice dosed every 4 days for 28 days


title: sKH1_NOVIRUSEtOH_2

source_name: control SKH-1 cells treated with vehicle ethanol, EtOH  replicate 2

value: SKH-1 cells

value: control SKH-1 cells


title: sKH1_NOVIRUS17BE2_2

source_name: control SKH-1 cells treated with E2 (estradiol) replicate 2


title: sKH1_NOVIRUS17BE2_1

source_name: control SKH-1 cells treated with E2 (estradiol) replicate 1


title: B-23

description: Mice dosed every 4 days for 28 days


title: sKH1_NOVIRUSEtOH_1

source_name: control SKH-1 cells treated with vehicle ethanol, EtOH  replicate 1


title: sKH1_CEBPAERE2_1

source_name: inducible version of CEBPA (C/EBPα-ER) in sKH1 cells treated with E2 (estradiol) replicate 1


title: sKH1_CEBPAERE2_2

source_name: inducible version of CEBPA (C/EBPα-ER) in sKH1 cells treated with E2 (estradiol) replicate 2


title: B-08

description: Mice dosed every 4 days for 28 days

value: Vehicle


title: B-15

description: Mice dosed every 4 days for 28 days


title: B-22

description: Mice dosed every 4 days for 28 days


title: B-21

description: Mice dosed every 4 days for 28 days


title: sKH1_CEBPAEREtOH_1

source_name: inducible version of CEBPA (C/EBPα-ER) in sKH1 cells treated with vehicle ethanol, EtOH replicate 1


title: B-13

description: Mice dosed every 4 days for 28 days


title: B-07

description: Mice dosed every 4 days for 28 days


title: B-14

description: Mice dosed every 4 days for 28 days


title: Kasumi1_RNAseq_1_Erplus

source_name: inducible version of CEBPA (C/EBPα-ER) in Kasumi-1 cells treated with E2 (estradiol) replicate 1


title: CEBPAKasumi1_E2_ChIPseq

library_source: genomic

data_processing:
Quality of reads was verified using FASTQC analysis
Sequences reads in fastq format were mapped onto the reference human genome version hg38, Genome Reference Consortium GRCh38. The Illumina reads were aligned to the human genome using Bowtie2
Reads that were uniquely aligned to chromosomal positions were retained and duplicate reads were removed from the aligned data using Picard tools.
The filtered aligned reads were used to generate density profiles using “genomeCoverageBed” function from bedtools
Genome_build: hg38
Supplementary_files_format_and_content: Wig

molecule: genomic DNA

source_name: CEBPA ChIP Kasumi1-1 cells treated with E2 (estradiol)

extract_protocol:
Each aliquot of 2x107 cells was resuspended in 600μl of sonication buffer (Tris-HCL pH8 25mM, NaCL 150mM, EDTA 2mM, Triton 100x 1%, SDS 0.25%, Protease inhibitior cocktail (PIC) 1x).  300 μl of nuclei in sonication buffer was placed in each polystyrene tube and sonicated at 75% amplitude, 26 cycles: 30s on and 30s off per cycle (Q800, Active Motif, USA).  Subsequently, 1.2ml of dilution buffer (Tris-HCL pH8 25mM, NaCL 150mM, EDTA 2mM, Triton 100x 1%, glycerol 7.5%, PIC 1x) was added to the pooled post sonication material.  This was divided equally between four immunoprecipitations (with 5% of input taken for validation).15 μl protein G beads (Diagenode, Belgium) were washed twice with 500 μl of 50mM citrate phosphate buffer and once with 100mM sodium phosphate).  2μg antibody (EVI-1, C50E12, Cell signalling, lot 3; or RUNX1, Ab23980, Abcam lot 144722) or 4µg antibody (C/EBPα, A2814 Santa Cruz) was added to 10 μl 100mM sodium phosphate, 0.5% BSA and incubated with protein G beads at 4°C for 1 hour.  Chromatin was then added to the protein G beads with antibody and returned to 4°C for 4 hours.  Unbound chromatin was separated from the beads by magnet and the attached beads were washed by buffer 1 (Tris HCL 20mM, NaCl 150mM, EDTA 2mM, Triton x100 1%, SDS 0.1%), twice with buffer 2 (Tris HCL 20mM, NaCl 500mM, EDTA 2mM, Triton x100 1%, SDS 0.1%), LiCL buffer (Tris HCL 10mM, LiCl 250mM, EDTA 1mM, NP40 0.5%, sodium deoxychlolate 0.5%) and finally twice with wash buffer 4 (Tris HCL pH8, 10mM, NaCl 50mM, EDTA 1mM).  The column was eluted twice with 50 μl buffer (NaHCO3 100mM and SDS 1%) and the eluant containing the chromatin was pooled.  Crosslinks were reversed by incubating the samples at 65°C overnight in 500mM NaCl, 500 μg/ml proteinase K.  DNA was purified by Ampure beads (Beckman Coulter, USA), as above, with the DNA eluted with 50 μl water.  Validation of the ChIP was performed by qPCR using a standard curve of genomic DNA from untreated SKH-1 cells (10ng/ μl followed by serial 1:5 dilutions).  The input material was diluted 1:5 with water and qPCR was performed as above with primers listed in SI.  Validation was analysed as a ratio of the qPCR signal from the ChIP material over the input.
Libraries for high throughput sequencing were prepared using the Tru-seq DNA sample preparation kit (Illumina, USA) or Kapa HyperPrep kit (Kapa biosystems, USA), as per manufacturer’s protocol.  18 cycles of PCR was performed and 200-350bp fragments were size selected by running the samples in an agarose gel.  Libraries were purified from the gel using a Minieluta Gel extraction kit (Qiagen, USA).  Libraries were validated by qPCR, with an analysis of the ChIP signal of a positive control region (e.g. PU.1 3H enhancer) over a negative control region (e.g. IVL).  Finally, libraries were quantified by Kapa library quantification kit (Kapa biosystems, USA) and run in a pool of four indexed libraries in one lane of a HiSeq 2500 (Illumina, USA) or 12 indexed libraries in one lane of a NextSeq 500 (Illumina, USA) using 50 cycle single-end reads.

value: vehicle E2

tag: antibody

value: C/EBPalpha (Santa Cruz, serial no.:A2814)


title: CEBPAKasumi1_ChIPseq

source_name: CEBPA ChIP control Kasumi1-1 cells treated with vehicle control-ethanol

value: vehicle control-ethanol


title: B-05

description: Mice dosed every 4 days for 28 days

source_name: Femur

value: Male


title: CEBPACEBPAER_Kasumi1_ChIPseq

source_name: CEBPA ChIP with inducible version of CEBPA (C/EBPα-ER) treated with vehicle control-ethanol

value: inducible version of CEBPA (C/EBPalpha-ER)


title: sKH1_CEBPAEREtOH_2

source_name: inducible version of CEBPA (C/EBPα-ER) in sKH1 cells treated with vehicle ethanol, EtOH replicate 2


title: CEBPA-SKHI-E2_ChIPseq

source_name: CEBPA ChIP sKH1-1 cells treated with E2 (estradiol)


title: Evi1-SKH1-E2_ChIPseq

source_name: RUNX1-EVI1 ChIP sKH1-1 cells treated with E2 (estradiol)

value: EVI-1 (Cell Signalling, serial no.:2593)


title: CEBPA-SKH1-CEBPA-ER_ChIPseq

source_name: CEBPA ChIP SKH1 cells with inducible version of CEBPA (C/EBPα-ER) treated with vehicle control-ethanol


title: CEBPA-SKH1_ChIPseq

source_name: CEBPA ChIP control sKH1 cells untreated treated with vehicle control-ethanol


title: Eto-Kasumi1_E2_ChIPseq

source_name: RUNX1-ETO ChIP Kasumi1-1 cells treated with E2 (estradiol)

value: ETO (Santa Cruz, serial no.:SC9737)


title: Evi1-SKH1_ChIPseq

source_name: RUNX1-EVI1 ChIP control sKH1 cells untreated treated with vehicle control-ethanol


title: CEBPA-SKH1-CEBPA-ER-E2_ChIPseq

source_name: CEBPA ChIP SKH1 cells with inducible version of CEBPA (C/EBPα-ER) treated with E2 (estradiol)


title: Eto-CEPBA-ER_ChIPseq

source_name: RUNX1-ETO ChIP with inducible version of CEBPA (C/EBPα-ER) treated with vehicle control-ethanol


title: Negative_3

city: Cambridge

first: Giovanni

last: Marsico

address: Robinson Way

institute: CRUK Cambridge Institute

description: PolyA RNA

data_processing:
RNA-seq: fastq raw sequencing files were preprocessed using cutadapt to remove sequencing adapters and low quality sequencing tails (options –q 10). Trimmed files were aligned to the human genome (GRCh37/hg19) using tophat2 and using the UCSC gtf file provided by Illumina iGenomes as an annotation file (http://support.illumina.com/sequencing/sequencing_software/igenome.html). Gene counts were calculated using htseq-count and the same gtf file. Differential expression analysis was done using the R package edgeR.
iCLIP-seq: identical reads were removed from the file, libraries were demultiplexed according to their 4-nucleotide patter sequence at the 5' end of each read (e.g., N3-GGTT-N2) and were then pre-processed with cutadapt to cut 3' sequencing adapters and low quality sequencing tails. Highly repetitive reads, i.e. those having at least 10 equal nucleotides (e.g., A{10,n}), were removed and reads aligned to the hg19 version of the human genome using bwa mem. After alignment, reads with MAPQ < 10 and those aligning to the same position while also having the same barcode were removed, as they constitute most likely PCR duplicates. Regions with signal above 10 were extracted, and regions closer than 30nt were merged into a single peak region. Merged peak regions less than 30nt in width were filtered out. Additionally, libraries were aligned to the transcriptome and processed: from filtered bwa-aligned bam files, only reads aligning with MAPQ >= 10 to expressed transcripts were retained. Expressed transcripts were considered as those having at least an average FPRM of 0.1 per condition (WT, mutant or negative) from the RNA-seq libraries, as assessed by cufflink isoform quantification. The remaining unfiltered reads were aligned to the expressed transcriptome using the software rsem. Coverage transcript files were calculated and normalized for the total estimated count in each iCLIP library, and peaks were called similarly to what done previously for the genome alignments. For peaks below 100 nt, the 100 base pairs around the middle of the peak were considered as binding regions, and sequences extracted. WT or mutant specific peaks were computer with the bedtools.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *.peaks.bed
Supplementary_files_format_and_content: *.norm.bedGraph
Supplementary_files_format_and_content: *_hg19_accepted_hits.nsort.htseq.txt: Raw counts.

source_name: Flp-In T-Rex 293

growth_protocol: Cells were grown to subconfluence in DMEM 10% FCS.

extract_protocol:
RNA extracted using TRI® reagent (Sigma-Aldrich) as per manufacturer's instructions. iCLIP performed as in Huppertz et al., Methods, 2014 with changes allowing cytoplasmic RNP isolation (Herdy et al., Nat. Immunol, 2012).
RNA-seq libraries were generated using the Illumina TruSeq Stranded total RNA kit (Illumina, cat #RS-122-2301) as per the manufacturer’s instructions. iCLIP libraries prepared as in Huppertz et al., Methods, 2014 with RT buffer allowing efficient quadruplex reverse transcription (Kwok et al., Nat. Methods, 2016). Sequenced using an Illumina NextSeq 500.

treatment_protocol: Protein expression induced with 0.1ug/ml Doxycycline.

value: Flp-In T-Rex 293

value: untreated


title: Negative_2

description: PolyA RNA


title: M244 DMSO

source_name: Melanoma cell line

value: Melanoma


title: Eto-Kasumi1_ChIPseq

source_name: RUNX1-ETO ChIP control Kasumi1-1 cells untreated  treated with vehicle control-ethanol

value: Kasumi-1 cells


title: Evi1-SKH1-CEBPA-ER-E2_ChIPseq

source_name: RUNX1-EVI1 ChIP with inducible version of CEBPA (C/EBPα-ER) treated with E2 (estradiol)


title: DDX3X_WT_4

description: PolyA RNA


title: murine hepatocytes (MMH-D3) H3K4me3

city: Wuppertal

first: Jan

last: Postberg

state: NRW

address: Heusnerstr. 40

country: Germany

institute: HELIOS University Hospital Wuppertal, Witten/Herdecke University

data_processing:
bcl2fastq2 v2.15.0 was used for demultiplexing
Fastqc 0.10.1 was used for read quality assessment
Cutadapt-1.2.1 was used for adaptor trimming.
Fastq files were uploaded onto the Galaxy server and converted using FASTQ Groomer
Read mapping was done using Bowtie2 with the ‘very sensitive end-to-end’ option.
The resulting BAM file was used for direct visualization of mapped reads in Geneious R9 software or further processed for comparative analyses using a DeepTools pipeline in Galaxy (1. bamCoverage [variable bin sizes, 50kbp among others] to generate coverage bigWig files for visualization in the UCSC genome browser (genome.ucsc.edu); 2. bigwigCompare with default options to normalize and compare two bigwig files and visualize the results in the UCSC genome browser.
Alternative color grading look-up tables (LUT) contained in WCIF ImageJ software were assigned to the UCSC genome browser greyscale dense coverage tracks for improved visualization.
Genome_build: Mus musculus genome mm9 or Homo sapiens hg38, respectively
Supplementary_files_format_and_content: bigwig format, files contain mapped reads on chromosomes Y, 15, 3, 1 for visualization of each investigated marker

source_name: murine hepatocytes (MMH-D3)

extract_protocol:
Briefly, lysates from cells crosslinked for 10 min in 1% formaldehyde were layered onto a 1.6 M sucrose cushion (1.6 M sucrose in 60 mM, KCl 15 mM NaCl, 5 mM MgCl2, 0.1 mM ethylene glycol tetraacetic acid (EGTA), 15 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 0.1 mM phenylmethanesulfonylfluoride (PMSF), 3.6 ng/mL aprotinin) and centrifuged. Nuclei were collected and portions corresponding to 50μg chromatin were sheared by ultrasonic treatment using a Bioruptor UCD-200 (Diagenode) and 30× [30 s ON/30 s OFF] at position ‘high’. Chromatin fragment size was controlled on agarose gels, and one of the chromatin aliquots was saved as input. For ChIP analyses, sheared chromatin samples were incubated with the respective antibody-of-interest in a rotator for 16 h at 4°C. Subsequently 20 μl protein G magnetic beads (Diagenode) were added and incubated for 4 hrs at 4°C rotating. Immunocomplexes attached to protein G magnetic beads were separated using a magnetic rack and washed repeatedly. To elute DNA fragments enriched by immunoprecipitation, immunocomplexes were incubated with elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, (pH 8.1)) for 30 min at 65°C on a shaker. Eluted immunocomplexes were treated with proteinase K overnight at 55°C. Antibodies used for IP were directed against HBxAg (mouse monoclonal antibody, clone 3F6-G10; Bio-Rad Laboratories Inc.), Pol2 (Active Motif, Carlsbad, CA, USA), H3K4me3 (Diagenode, Liège, Belgium), H3K4me1 (Diagenode, Liège, Belgium), H3K9me3 (Diagenode, Liège, Belgium), H3K9ac (Diagenode, Liège, Belgium), H3K27me3 (Diagenode, Liège, Belgium), H3K27ac (Diagenode, Liège, Belgium), H3K36me3 (Diagenode, Liège, Belgium) or H3K36ac (Diagenode, Liège, Belgium). DNA associated with enriched immunocomplexes was purified by phenol:chloroform:isoamylic alcohol extraction and precipitated with isopropylic alcohol.
Illumina-compatible libraries were prepared using the MicroPlex Library Preparation kit v2 (Diagenode, Liège, Belgium). Massive parallel sequencing was performed as described below.
ChIP-seq was performed with the paired-end or, alternatively, with the single read option (42nt paired-end in mmu experiments/50nt single-end in hsa experiments) and the Illumina NextSeq 500 High Output Kit

tag: tumour stage

value: non-transformed

tag: source

value: liver


title: CEBPACEBPAER_E2_Kasumi1_ChIPseq

source_name: CEBPA ChIP with inducible version of CEBPA (C/EBPα-ER) treated with E2 (estradiol)


title: DDX3X_WT_2

description: PolyA RNA


title: DDX3X_mRG_4

description: PolyA RNA


title: DDX3X_mRG_3

description: PolyA RNA


title: DDX3X_WT_1

description: PolyA RNA


title: DDX3X_mRG_1

description: PolyA RNA


title: Eto-CEBPA-ER-E2_ChIPseq

source_name: RUNX1-ETO ChIP with inducible version of CEBPA (C/EBPα-ER) treated with E2 (estradiol)


title: WT3

data_processing:
Library strategy: iCLIP-Seq
RNA-seq: fastq raw sequencing files were preprocessed using cutadapt to remove sequencing adapters and low quality sequencing tails (options –q 10). Trimmed files were aligned to the human genome (GRCh37/hg19) using tophat2 and using the UCSC gtf file provided by Illumina iGenomes as an annotation file (http://support.illumina.com/sequencing/sequencing_software/igenome.html). Gene counts were calculated using htseq-count and the same gtf file. Differential expression analysis was done using the R package edgeR.
iCLIP-seq: identical reads were removed from the file, libraries were demultiplexed according to their 4-nucleotide patter sequence at the 5' end of each read (e.g., N3-GGTT-N2) and were then pre-processed with cutadapt to cut 3' sequencing adapters and low quality sequencing tails. Highly repetitive reads, i.e. those having at least 10 equal nucleotides (e.g., A{10,n}), were removed and reads aligned to the hg19 version of the human genome using bwa mem. After alignment, reads with MAPQ < 10 and those aligning to the same position while also having the same barcode were removed, as they constitute most likely PCR duplicates. Regions with signal above 10 were extracted, and regions closer than 30nt were merged into a single peak region. Merged peak regions less than 30nt in width were filtered out. Additionally, libraries were aligned to the transcriptome and processed: from filtered bwa-aligned bam files, only reads aligning with MAPQ >= 10 to expressed transcripts were retained. Expressed transcripts were considered as those having at least an average FPRM of 0.1 per condition (WT, mutant or negative) from the RNA-seq libraries, as assessed by cufflink isoform quantification. The remaining unfiltered reads were aligned to the expressed transcriptome using the software rsem. Coverage transcript files were calculated and normalized for the total estimated count in each iCLIP library, and peaks were called similarly to what done previously for the genome alignments. For peaks below 100 nt, the 100 base pairs around the middle of the peak were considered as binding regions, and sequences extracted. WT or mutant specific peaks were computer with the bedtools.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *.peaks.bed
Supplementary_files_format_and_content: *.norm.bedGraph
Supplementary_files_format_and_content: *_hg19_accepted_hits.nsort.htseq.txt: Raw counts.

molecule: cytoplasmic RNA


title: WT1


title: Evi1-SKH1-CEBPA-ER_ChIPseq

source_name: RUNX1-EVI1 ChIP with inducible version of CEBPA (C/EBPα-ER) treated with vehicle control-ethanol


title: Negative_1

description: PolyA RNA


title: mRG3


title: DDX3X_WT_3

description: PolyA RNA


title: mRG1


title: All_reads

value: merged wt and mutant DDX3X


title: RNA-DHX9-3

description:
Total RNA, Ribosome depleted
RNA-seq

data_processing:
RNA-seq libraries were trimmed using cutadapt. The remaining reads were aligned to the GRCh38 human genome using the rsem-calculate-expression of RSEM
For all analyses the version 26 of the human genome transcript annotation from Gencode was used. For each experiment, the transcriptome was defined by considering transcripts with an expression above 1 TPM (Transcript Per Million) and isoform representation percentage above 5%.
22,911 transcripts representing the union transcriptome of all performed RNA-seq experiments were then considered for all following analyses, and the corresponding RPF libraries were aligned to it using RSEM. Only RPF reads in the size range 24-32 were considered for analysis.
Isoforms quantifications for both RNA-seq and RPF libraries were obtained in forms of estimated counts and TPM during the alignment procedure. Transcript coverage was calculated using RSEM command rsem-bam2wig and rsem-bam2readdepth, which both take into account multi-mapping reads and proportionally assign multiple assignments, a crucial step for processing short reads libraries.
For iCLIP analysis, refer to associated publicatoon.
Genome_build: GRCh38

source_name: ATCC Cell Lines

growth_protocol: HeLa cells were cultured in Dulbecco’s modified Eagle Medium (Life Technology) supplemented with 10% FBS (Life Technology).

extract_protocol:
Total RNA and ribosome-protected fragments (RPFs) were isolated using the TruSeq Ribo Profile Mammalian Kit (Illumina) following the manufacturer’s intructions with some modifications. For RPFs purification, lysates were treated with 5 U TruSeq Ribo Profile Nuclease per A260 of lysate. RPFs were isolated using Illustra MicroSpin S-400 HR Columns (GE Healthcare, 27- 5140-01). rRNAs were removed using the Ribo-zero Magnetic Gold kit (Illumina, MRZG12324). After PAGE purification (15% urea-polyacrylamide gel to select fragments ~28-30 nt in length), end-repair, 3’-adapter ligation, reverse transcription (EpiScript reverse transcriptase) and cDNA circularisation (CircLigase, Epicentre), RPFs were amplified using 9 PCR cycles using Phusion polymerase (NEB). For parallel RNA-seq, total RNA was processed similarly, but excluding the nuclease digestion and S-400 columns purification steps. Before the library preparation steps, total RNA samples were heat-fragmented (94°C for 25 minutes). PCR amplicons were purified on a 8% native polyacrylamide gel excising bands corresponding to ~70-80 nt and ~80-100 nt for RPF and total RNA samples respectively. Libraries were sequenced on a NextSeq 500 using 75-nt single-read sequencing runs.  For iCLIP, refer to associated publication.
TruSeq Ribo Profile Mammalian Kit (Illumina)
RNA-Seq, Ribo-Seq, iCLIP-seq

treatment_protocol: For siRNA transfection, 5 × 10^6 HeLa cells per 15 cm dish were cultured overnight and treated with 27 μL DharmaFECT 1 Transfection Reagent (Dharmacon) and treated with 90 nM indicated siRNA in 1.8 mL OptiMEM media.DHX9 and DHX36 siRNA oligoes were transfected at equimolar concentrations. A pool of four non-targeting siRNAs (ON-TARGETplus Non-targeting Control Pool, Dharmacon) was used as control. The cells were then expended for 48 h. For iCLIP, refer to associated publication.

value: HeLa

value: DHX9 siRNA


title: RNA_DHX9_1

description:
Total RNA, Ribosome depleted
RNA-seq


title: RNA_DHX36_1

description:
Total RNA, Ribosome depleted
RNA-seq

value: DHX36 siRNA


title: RNA_DHX36_2

description:
Total RNA, Ribosome depleted
RNA-seq


title: RNA-sc-4

description:
Total RNA, Ribosome depleted
RNA-seq

value: scramble siRNA


title: RNA-DHX36-3

description:
Total RNA, Ribosome depleted
RNA-seq


title: RNA_sc_2

description:
Total RNA, Ribosome depleted
RNA-seq


title: DDX3X_mRG_2

description: PolyA RNA


title: WT2


title: RNA-NT-2

description:
Total RNA, Ribosome depleted
RNA-seq


title: RPF-DHX9-3

description:
Ribosome protected RNA fragments
Ribo-seq


title: RNA-NT-1

description:
Ribosome protected RNA fragments
RNA-seq


title: RPF_DHX36_2


title: RPF-DHX36-3


title: RNA-sc-3

description:
Total RNA, Ribosome depleted
RNA-seq


title: RPF_DHX9_2


title: RPF_DHX36_1


title: RPF-sc-3


title: RPF-sc-4


title: RPF_sc_2


title: RPF_DHX9_1


title: mRG2


title: RNA_DHX9_2

description:
Total RNA, Ribosome depleted
RNA-seq


title: RPF-NT-1


title: iCLIP-DHX9-1

description:
RNA crosslinked to DHX9
iCLIP-Seq


title: iCLIP-DHX9-2

description:
RNA crosslinked to DHX9
iCLIP-Seq


title: RPF-NT-2


title: VRTN_siRNA_rep3

city: Jerusalem

first: Mordecai

last: Peretz

address: The Edmond J. Safra Campus

country: Israel

institute: Hebrew University of Jerusalem

description:
eGFP gene under Rex1 promoter.
Processed data file: rawCounts.csv

data_processing:
Reads were mapped to the reference genome using STAR 2.4.0.1 software.
Reads were annotated using HTSeq 0.9.1 software.
Genome_build: GRCh38 reference genome
Supplementary_files_format_and_content: rawCounts.csv: Comma-separated values (csv) file contains raw transcript counts for each sample.

source_name: embryonic stem cells, VRTN siRNA

growth_protocol: Cells were cultured at 37 C and 5% CO2 in feeder-free conditions on Matrigel-coated plates (Corning) in mTeSR1 (STEMCELL Technologies).

extract_protocol:
Cells were harvested 48 hours after transfection of siRNAs for SALL4 and VRTN and 72 hours for DSCC1 and SEPHS1. Total RNA was isolated from three independent biological replicates with RNeasy Mini or Micro Kit (QIAGEN).
mRNA fraction of total RNA was enriched by pulldown of poly(A)-RNA. RNA sequencing libraries were generated using SENSE Total RNA-Seq Library Prep Kit (LEXOGEN) according to manufacturer’s protocol and sequenced using Illumina NextSeq 500 with 80 bp single-end reads.

treatment_protocol: All genes were targeted with commercial pooled siRNAs to increase the specificity of the knockdown. esiRNAs for SALL4, DSCC1 and SEPHS1 and the control esiRNA for Renilla Luciferase were obtained from Sigma. siRNA for VRTN was obtained from GE Healthcare Dharmacon. Briefly, 30-50 nM siRNA was mixed with 1.4 µl of DharmaFECT 1 transfection reagent (GE Healthcare Dharmacon) in Opti-MEM I reduced serum media (Thermo Fisher Scientific) for 30 minutes. The transfection mix was then added to mTeSR 1 medium with ~30-50k hESCs and the cells were plated on a Matrigel-coated well of a 12-well plate. The cells were subsequently grown for 3-4 days and the mTeSR 1 medium was replaced every 24 hours.

tag: source cell line

value: embryonic stem cells

tag: genotype/variation

tag: sirna

value: VRTN


title: SEPHS1_siRNA_rep1

source_name: embryonic stem cells, SEPHS1 siRNA


title: SEPHS1_siRNA_rep3


title: DSCC1_siRNA_rep3

source_name: embryonic stem cells, DSCC1 siRNA


title: VRTN_siRNA_rep2


title: VRTN_siRNA_rep1


title: DSCC1_siRNA_rep2


title: DSCC1_siRNA_rep1


title: SALL4_siRNA_rep3

source_name: embryonic stem cells, SALL4 siRNA


title: SALL4_siRNA_rep1


title: mRNA-seq HepG2 H1.3

source_name: human hepatocytes (HepG2 H1.3)

extract_protocol:
Prior to gene expression analyses, total RNA was isolated from at least three biological replicates for each experiment using Trizol reagent (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s recommendations. RNA integrity was validated using an Agilent Bioanalyzer 2100 with the Agilent RNA 6000 Nano Kit for microcapillary electrophoresis (Agilent Technologies, Santa Clara, CA, USA). We determined RNA transcription profiles of several hepatocyte cell populations (HepaRG, HepG2.2.15, HepG2 H1.3). For mRNA enrichment we used the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and then the NEBNext Ultra RNA Kit (NEB) for Illumina-compatible library preparation upon manufacturer’s recommendations.
Illumina-compatible libraries were prepared using the NEB NExt Library Preparation kit (New England Biolabs) in combination with the poly-[A] enrichment module. Massive parallel sequencing was performed as described below.
mRNA-seq was performed with the single-end read option (50nt paired-end) and the Illumina NextSeq 500 High Output Kit

value: transformed


title: SALL4_siRNA_rep2


title: mRNA-seq HepG2.2.15

source_name: human hepatocytes (HepG2.2.15)


title: Control_siRNA_rep1

source_name: embryonic stem cells, Control siRNA

value: Control


title: mRNA-seq HepaRG t2

source_name: human hepatocytes (HepaRG)

value: primary cell-like


title: Luciferase_siRNA_rep1

source_name: embryonic stem cells, Luciferase siRNA

value: Luciferase


title: Control_siRNA_rep2


title: Luciferase_siRNA_rep2


title: 4C_cccDNA-HepaRG

data_processing:
Library strategy: 4C-Seq
bcl2fastq2 v2.15.0 was used for demultiplexing
Fastqc 0.10.1 was used for read quality assessment
Cutadapt-1.2.1 was used for adaptor trimming.
Fastq files were uploaded onto the Galaxy server and converted using FASTQ Groomer
Read mapping was done using Bowtie2 with the ‘very sensitive end-to-end’ option.
The resulting BAM file was used for direct visualization of mapped reads in Geneious R9 software or further processed for comparative analyses using a DeepTools pipeline in Galaxy (1. bamCoverage [variable bin sizes, 50kbp among others] to generate coverage bigWig files for visualization in the UCSC genome browser (genome.ucsc.edu); 2. bigwigCompare with default options to normalize and compare two bigwig files and visualize the results in the UCSC genome browser.
Alternative color grading look-up tables (LUT) contained in WCIF ImageJ software were assigned to the UCSC genome browser greyscale dense coverage tracks for improved visualization.
Genome_build: Mus musculus genome mm9 or Homo sapiens hg38, respectively
Supplementary_files_format_and_content: bigwig format, files contain mapped reads on chromosomes Y, 15, 3, 1 for visualization of each investigated marker

extract_protocol:
In order to select a restriction strategy applicable for multiple HBV genotypes, we performed alignments using ClustalW included in molecular evolutionary genetics analysis (MEGA) 4.1. The genotype HBV D3, which is replicated by HepG2.2.15 cells and used for the infection of HepaRG cells, was verified by PCR using primers giving rise to an approx. 590 bp amplicon (depending on the genotype) as described above and Sanger sequencing of amplicons cloned into pGEM-T easy (Promega) via the standard T7 sequencing primer. 4C targeting on HBV cccDNA (verified genotype D3) as bait was conducted as described previously with slight modifications. Briefly, interacting DNA segments were crosslinked using 1% formaldehyde and nuclei were isolated using a lysis buffer containing 0.4% Tergitol (NP-40 substitute) followed by incubation at 37°C in presence of 0.3% SDS. Prior to the primary digestion step SDS was sequestered using 2% Triton X-100, and subsequently SpeI (NEB, 600U, 24 hrs) was added. Digestion efficiencies were quantified by qPCR using a Corbett Rotor-Gene 6000 qPCR device and Quantitect SYBR Green Master Mix (Qiagen, Hilden, Germany) and primers covering the SpeI restriction site within the cccDNA normalized to a non-digested region. Digested samples were purified, diluted and then proximity-ligated at 16°C using T4 DNA ligase (Roche). For 4C library construction the crosslink was reversed. Samples were subjected to digestion with HaeIII (NEB, 20 U per µL, 24 hrs) subsequently, followed by a second ligation. Using specific primer pairs (Primer A: 5’- cccactgtttggctttcag-3’/Primer B: 5’-gggaaagccctacgaaccac-3’, or respectively, Primer C: 5’- gtttctcctggctcagtttac-3’/Primer D: 5’- gtttctcytggctcagtttac-3’) adjacent to the respective ligation sites, genomic sequences associated with HBV cccDNA were amplified. From these amplicons Illumina-compatible libraries were made using the MicroPlex Library Preparation kit v2 (Diagenode, Liège, Belgium). Massive parallel sequencing was performed as described below.
4C targeting on HBV cccDNA (verified genotype D3) as bait was conducted as described previously (42,43) with slight modifications (Figure S2C). Briefly, interacting DNA segments were crosslinked using 1% formaldehyde and nuclei were isolated using a lysis buffer containing 0.4% Tergitol (NP-40 substitute) followed by incubation at 37°C in presence of 0.3% SDS. Prior to the primary digestion step SDS was sequestered using 2% Triton X-100, and subsequently SpeI (NEB, 600U, 24 hrs) was added. Digestion efficiencies were quantified by qPCR using a Corbett Rotor-Gene 6000 qPCR device and Quantitect SYBR Green Master Mix (Qiagen, Hilden, Germany) and primers covering the SpeI restriction site within the cccDNA normalized to a non-digested region. Digested samples were purified, diluted and then proximity-ligated at 16°C using T4 DNA ligase (Roche). For 4C library construction the crosslink was reversed. Samples were subjected to digestion with HaeIII (NEB, 20 U per µL, 24 hrs) subsequently, followed by a second ligation. Using specific primer pairs (Primer A: 5’- cccactgtttggctttcag-3’/Primer B: 5’-gggaaagccctacgaaccac-3’, or respectively, Primer C: 5’- gtttctcctggctcagtttac-3’/Primer D: 5’- gtttctcytggctcagtttac-3’) adjacent to the respective ligation sites, genomic sequences associated with HBV cccDNA were amplified. From these amplicons Illumina-compatible libraries were made using the MicroPlex Library Preparation kit v2 (Diagenode, Liège, Belgium). Massive parallel sequencing was performed as described below.
4C-seq was performed with the paired-end read option (75nt paired-end) and the Illumina NextSeq 500 High Output Kit


title: 4C_cccDNA-HepG2 H1.3


title: mRNA-seq HepaRG t0


title: 4C_cccDNA-HepG2.2.15


title: mRNA-seq HepaRG t1


title: human hepatocytes (HepaRG) H3K4me3


title: human hepatocytes (HepG2 H1.3) H3K4me3


title: SEPHS1_siRNA_rep2


title: human hepatocytes (HepG2 H1.3) H3K36me3


title: human hepatocytes (HepaRG) H3K36me3


title: human hepatocytes (HepG2.2.15) H3K4me3


title: human hepatocytes (HepaRG) HBx

value: HBx


title: human hepatocytes (HepG2.2.15) HBx


title: human hepatocytes (HepaRG) Pol2


title: human hepatocytes (HepG2.2.15) H3K36me3


title: human hepatocytes (HepG2.2.15) Pol2


title: human hepatocytes (HepG2 H1.3) HBx


title: mRNA-seq HepaRG t3


title: Sample 3 GFPneg

city: Paris

first: Silvia

last: Fre

address: 26 rue d’Ulm

department: Genetics and Developmental Biology

country: France

institute: Institut Curie

description: GFP negative (replicate 3)

data_processing:
Quality of reads was assessed for each sample using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Raw reads were trimmed to remove the first three bases of the first read using Cutadapt tool.
Fastq files were aligned to the reference mouse genome mm10 with TopHat2 (-p 24 -r 150 -g 2 --library-type fr-secondstrand). We removed reads mapping to multiple locations.
We used HTSeq to obtain the number of reads associated to each gene in the Gencode database (restricted to protein-coding genes, antisense and lincRNAs).
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files contain read counts for a single sample.

source_name: Pubertal mouse mammary basal cells

extract_protocol:
Total RNA was extracted from sorted mammary cells using the RNeasy Extraction kit (QIAGEN).
RNA libraries were prepared for sequencing using standard Illumina protocols and sequenced on paired end 2x75b run on the Illumina HiSeq4000

treatment_protocol: Mice were induced with a single dose of tamoxifen at postnatal day P21 and mammary cells were isolated 72h later (P24).

value: Mammary basal cells

tag: developmental stage

value: Postnatal day 24


title: Sample 3 GFPpos

description: GFP positive (replicate 3)

tag: strain


title: Sample 2 GFPneg

description: GFP negative (replicate 2)


title: Sample 1 GFPpos

description: GFP positive (replicate 1)


title: Sample 1 GFPneg

description: GFP negative (replicate 1)


title: Sample 2 GFPpos

description: GFP positive (replicate 2)


title: nr48

city: Perth

first: Sulev

last: Koks

state: Western Australia

address: 90 South Street

department: Centre for Molecular Medicine and Innovative Therapeutics

country: Australia

institute: Murdoch University

data_processing:
Color-space base calling
Mapping, alignment with Lifescope
Lifescope transcriptome workflow
Genome_build: hg19

source_name: blood

growth_protocol: sample from the blood

extract_protocol:
Blood samples were collected with the Tempus Blood RNA Tubes. The samples were stored at – 20 °C until RNA extraction. For total RNA extraction the combination of TRIzol Reagent and RNeasy Mini Kit was used.
WT RNA-seq libraries were prepared for 48 RNA samples. 50 ng of each Globin Clear kit treated total RNA sample was taken as library input. The rest of the RNA material is stored at – 80 °C. For library preparation the NuGen Ovation RNA seq V2 kit together with 5500 Series Fragment Library Core Kit was used according to manufacturer’s protocol.

treatment_protocol: smokers, former-smokers and never-smokers

tag: smokingstatus

value: NEVER

tag: Sex

value: male

tag: eversmoked

value: no

tag: neversmoked

value: yes

tag: yearssmoked

tag: height

tag: weight

tag: waist

tag: hip

tag: barcode

value: BC 16


title: nr46

value: FORMER

tag: age

value: BC 14


title: nr41

value: CURRENTLY

value: BC 9


title: nr39

value: BC 7


title: nr37

value: BC 5


title: nr45

value: female

value: BC 13


title: nr42

value: BC 10


title: nr43

value: BC 11


title: nr40

value: BC 8


title: nr47

value: BC 15


title: human hepatocytes (HepG2 H1.3) Pol2

tag: tumour stage

value: transformed

tag: source

value: liver

tag: antibody


title: nr35

value: male

value: no

value: BC 3


title: nr32

value: FORMER

value: yes

value: BC 16


title: nr38

value: CURRENTLY

value: BC 6


title: nr31


title: nr33

value: NEVER

value: BC 1


title: nr36

value: female

value: BC 4


title: nr29

value: BC 13


title: nr44

value: BC 12


title: nr30

value: BC 14


title: nr22


title: nr25

value: BC 9


title: nr24

value: BC 8


title: nr23

value: BC 7


title: nr27

value: male

value: no

value: BC 11


title: nr21

value: yes

value: BC 5


title: nr34

value: FORMER

value: BC 2


title: murine hepatocytes (MMH-D3) H3K27ac

value: non-transformed

value: liver


title: nr19

value: NEVER

value: female

value: BC 3


title: nr20

value: BC 4


title: nr28

value: BC 12


title: nr12


title: nr17

value: CURRENTLY

value: BC 1


title: nr18


title: nr15

value: BC 15


title: nr10


title: nr9


title: nr16

value: BC 16


title: nr11

value: male

value: no


title: nr8

value: yes


title: nr7


title: nr13


title: nr6


title: nr3


title: nr2


title: nr1


title: nr26

value: BC 10


title: nr5


title: nr14

value: FORMER


title: C18:0 CoA treatment replicate 3

city: Antwerpen

first: Gerrit

last: Beemster

state: Antwerpen

address: Groenenborgerlaan 171

department: Biology

country: Belgium

institute: University of Antwerp

description:
Ro6_ATTACTCG-TAATCTTA_L006
Barcode: ATTACTCG-TAATCTTA

data_processing:
The base-calling happens in the Illumina software that controls the sequencer and the alignment (as well as the QC and read counting) was done with CLC Genomics workbench using default settings.
Transcriptome analysis was performed by means of CLC Genomics Workbench v.6 using the Arabidopsis thaliana reference sequence (Tair10). The expression values were normalized using quantile normalization and pair-wise statistical analyses comparing the treatments performed using FDR-corrected p-values based on Baggerley’s test (Baggerly, et al., 2003).
Genome_build: Tair10
Supplementary_files_format_and_content: normalized expression values

organism: Arabidopsis thaliana

source_name: In-vitro grown seedlings

growth_protocol: Wild-type seeds were sown in 24-well plates containing half-strength liquid MS medium + 0.5% sucrose and stratified for 48 hours at 4°C and germinated at 21°C day / 18°C night with a photoperiod of 16 h light (150 μmol m−2 s−1) and 8 h dark.

extract_protocol:
Total RNA was extracted using the NucleoSpin® RNA Plant kit from Macherey-Nagel according to the manufacturer's  instructions. Subsequently, contaminating DNA was removed using the Turbo DNA-free kit from Ambion according to the manufacturer's instructions.
RNA quality and integrity was assessed on a QIAxcel platform (Qiagen, Hilden, Germany). The TruSeq® Stranded mRNA sample preparation 96 rcx kit (Illumina™) following the low sample protocol according to Illumina™ guidelines was used to construct the library using 2.5 µg of total RNA. Adaptors were ligated in 12-plex formations and the libraries were quantified using PicoGreen® (Life Technologies™). Groups of 12 samples were pooled at equal concentrations to create the eight pools. The Kapa SYBR® FAST universal qPCR kit (Kapa Biosystems™) for Illumina™ sequencing was used to quantify the number of the amplifiable molecules in the pools and the Bioanalyzer® (Agilent Technologies™) to determine the average fragment size of our pools and optimization of the flow cell clustering. An illumina HiSeq 1500 sequencer was used in the high throughput mode using the TruSeq® SBS Kit in a 50-cycle pair-end run.

treatment_protocol: At day 14, seedlings were treated with different Acyl-CoAs (C18:0, C18:1 or C16:0) at a final concentration of 0.1% in 0.01% Pluronic F68.  For control treatmens plants were exposed to 0.01% Pluronic F68. After 90 minutes whole seedlings were collected and immediately frozen in liquid nitrogen.

tag: bbch scale

tag: tissue

value: whole seedlings

tag: plants per pool

tag: treatment

value: C18:0 CoA


title: C18:0 CoA treatment replicate 1

description:
Ro4_ATTACTCG-GGCTCTGA_L004
Barcode: ATTACTCG-GGCTCTGA


title: Control replicate 1

description:
Ro1_ATTACTCG-TATAGCCT_L001
Barcode: ATTACTCG-TATAGCCT

value: Control


title: C18:1 CoA treatment replicate 1

description:
Ro7_ATTACTCG-CAGGACGT_L007
Barcode: ATTACTCG-CAGGACGT

value: C18:1 CoA


title: C18:0 CoA treatment replicate 2

description:
Ro5_ATTACTCG-AGGCGAAG_L005
Barcode: ATTACTCG-AGGCGAAG


title: C16:0 CoA treatment replicate 1

description:
Ro10_TCCGGAGA-ATAGAGGC_L002
Barcode: TCCGGAGA-ATAGAGGC

value: C16:0 CoA


title: Control replicate 3

description:
Ro3_ATTACTCG-CCTATCCT_L003
Barcode: ATTACTCG-CCTATCCT


title: C16:0 CoA treatment replicate 2

description:
Ro11_TCCGGAGA-CCTATCCT_L003
Barcode: TCCGGAGA-CCTATCCT


title: D18-3_082

status: Public on Apr 29 2018

city: Daejeon

first: Ji-Hwan

last: Park

address: 125 Gwahak-ro, Yuseong-gu

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology (KRIBB)

data_processing:
Illumina CASAVA v1.8.2 software was used for basecalling.
We trimmed the adapter sequences (TruSeq™ index adapter with option “-a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC”, and the reverse complementary sequence of TruSeq™ universal adapter with the option “-a AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT”) by applying the cutadapter software
The remaining reads were then aligned to the Arabidopsis thaliana genome (TAIR10) with TopHat by allowing two mismatches and up to 20 different alignments (default). The aligned reads were assembled into genes by calculating the fragments per kilobase per million mapped reads (FPKMs) with Cufflinks.
After obtaining FPKM values, log2(FPKM+1) values of genes were normalized by quantile normalization
Genome_build: Arabidopsis_thaliana.TAIR10.29
Supplementary_files_format_and_content: tab-delimited text files including log2(FPKM+1) values after quantile normalization for each sample

molecule: polyA RNA

source_name: Arabidopsis thaliana leaf, 18 days

growth_protocol: Arabidopsis thaliana ecotype Columbia (Col) was the parent strain for all mutants used in this study. The T-DNA insertion lines of NAC genes were obtained from the SALK T-DNA insertion collection, SAIL collection, or GABI-KAT collection. Plants used for most of the experiments were grown in an environmentally controlled growth room under a 16-hr-light/8-hr-dark cycle (photon flux density 100 µmol m-2s-1) at 22°C.

extract_protocol:
Total RNAs were prepared from the leaves of anac017, anac082 and anac090 mutants and WT (n=3 for each). The RNA integrity of the samples were confirmed (RNA integrity number ≥7.8) by using the Agilent Technologies 2100 BioAnalyzer.
According to the manufacturer’s instructions, Poly(A) mRNA was isolated from the RNA (2 µg) and fragmented by using Illumina Truseq™ Stranded mRNA LT Sample Prep Kit with poly-T oligo-attached magnetic beads. After the reverse transcription of the RNA fragments by using Superscript II reverse transcriptase (Life Technologies, USA), the strand-specific cDNA libraries were constructed with adaptor-ligation and sequenced by Illumina HiSeq 2500 system.

tag: ecotype

value: Columbia (Col)

tag: genotype

value: anac082 mutant

value: leaf


title: D18-1_082


title: D18-2_017

value: anac017 mutant


title: D18-2_090

value: anac090 mutant


title: D18-2_082


title: D18-3_090


title: D18-1_017


title: C18:1 CoA treatment replicate 2

description:
Ro8_ATTACTCG-GTACTGAC_L008
Barcode: ATTACTCG-GTACTGAC


title: Control replicate 2

description:
Ro2_ATTACTCG-ATAGAGGC_L002
Barcode: ATTACTCG-ATAGAGGC


title: D18-1_090


title: D18-2_Col

value: Wild type


title: BT50/T8

city: Changsha

first: Lu

last: Xiaopeng

address: 1 Nongda Rad.

country: China

institute: Hunan Agricultural University

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Citrus  sinensis  whole genome
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Gene IDs in the processed data file can be accessed at: The Public Genome Database of orange  http://citrus.hzau.edu.cn/orange/
Genome_build: Citrus  sinensis
Supplementary_files_format_and_content: xls files include RPKM values for each sample

organism: Citrus sinensis

source_name: fruits

extract_protocol:
Endocarps were isolated, flash frozen in liquid nitrogen, and RNA was extracted using Trizol reagent.    NEBNext Poly（A）mRNA Magnetic Isolation Module（NEB, E7490）was used to enrich mRNA  for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: cultivar

value: Citrus sinensis L. Osbeck 'Bingtangcheng'

value: endocarps

tag: development stage

value: 50 days after full bloom


title: DH50/T7

value: Citrus sinensis L. Osbeck 'Dahongtiancheng'


title: DH70/T5

value: 70 days after full bloom


title: BT70/T6


title: D18-1_Col


title: C16:0 CoA treatment replicate 3

description:
Ro12_TCCGGAGA-GGCTCTGA_L004
Barcode: TCCGGAGA-GGCTCTGA


title: C18:1 CoA treatment replicate 3

description:
Ro9_TCCGGAGA-TATAGCCT_L001
Barcode: TCCGGAGA-TATAGCCT


title: D18-3_017


title: D18-3_Col


title: ireA- - TN rep2

status: Public on Apr 28 2018

city: Cd de Mexico

first: Roberto

last: Coria

state: Cd de Mexico

address: Apartado Postal 70-600

department: Molecular Genetics

country: Mexico

institute: Instituto Fisiologia Celular

data_processing:
Foreach condition and replica: tophat -p 16 gtf_file -o tophat_outdir fastq file. Tophat ve5rsion 2.1.0
Foreach tophat_outdir: cufflinks -p 16 -o cufflinks_outdir tophat_outdir/accepted_bam_file. Cufflinks version 2.2.1
cuffmerge -g genes.gtf -s dicty.fa -p 16 assemblies_file
For each replica: cuffdiff to calculate differential expression.
Genome_build: AX4 strain
Supplementary_files_format_and_content: processed.xls containing differentially expressed genes.

organism: Dictyostelium discoideum

growth_protocol: Dictyostelium discoideum cells were grown at 22oC. Axenic growth was performed in HL5 media supplemented with glucose 1% and penicillin–streptomycin (10 000 U /ml penicillin and 10 000 mg/ml streptomycin)

extract_protocol:
Cells were centrifuged and wash and total RNA was extracted with standard Trizol protocol
cDNA libraries were prepared using the TruSeq Sample Preparation v2 kit (Illumina) according to the manufacture's recomendations. Poly-A containing mRNA was purified from 1ug of total RNA, fragmented. Following fragmentation cDNA was synthesized. cDNA fragments were end repaired, and a single ‘A’ base was added (to the 3’ end of the blunt fragments), afterwards barcoded (indexed) adapters were ligated to each sample. The products were purified and enriched with a 15 cycles PCR to obtain the final cDNA libraries.

treatment_protocol: Axenically growing cells at exponential phase were adjusted a 1x10^6 cells/ml density and treated for 16hrs with 2ug/ml of TN or DMSO in HL5

value: ireA-

value: TN

tag: parental strain

tag: growth condition

value: Axenic

tag: phase of growth

value: Exponential


title: ireA- rep1

source_name: Axenically growing cells at exponential phase


title: WT rep1

value: WT


title: WT-TN rep2


title: WT rep2


title: ireA- - TN rep1


title: ireA- rep2


title: WT-TN rep1


title: Whole organism, Marshall population, 5 ppt treatment, replicate 5

city: Hayward

first: Tyler

middle: G

last: Evans

state: California

address: 25800 Carlos Bee Blvd

department: Biological Sciences

institute: California State University East Bay

description:
Sample 35
TE31C_S44_L005_R1

data_processing:
Adapter sequences (NEB Illumina Universal Adapters) were removed and quality trimming was performed using the following parameters: quality score 0.05 (Phred; Ewing & Green 1998; Ewing et al. 1998), number of ambiguous nucleotides < 2 on ends, and reads shorter than 20 bp were removed.
Sequences from each sample were mapped to a de novo transcriptome for O. lurida using CLC Genomics Workbench.
Raw count data exported from CLC Genomics were filtered, normalized, log-transformed, and statistically analyzed using default parameters of the DESeq2 package (Love et al. 2014) in R version 3.3.2 (R Development Core Team, 2011).
A generalized linear model was fit to the expression data to assess main effects of salinity and population, as well as an interaction between these factors. Contigs with Benjamini-Hochberg false discovery rate corrected p-values <0.05 were considered significantly differentially expressed.
Genome_build: Sequences from each sample were mapped to a de novo transcriptome for O. lurida using CLC Genomics Workbench (GEO O.lurida transcriptome.txt)
Supplementary_files_format_and_content: abundance measurements

organism: Ostrea lurida

source_name: Whole organism_Marshall population_5 ppt treatment

growth_protocol: All experiments were performed on second generation progeny of oysters collected from three sites within Northern California estuaries: (1) Loch Lomond and (2) Oyster Point in the San Francisco Bay, and (3) Marshall in Tomales Bay. Adults collected from each population (i.e. broodstock) were spawned in the laboratory and resulting first- and second-generation oysters raised under common garden conditions in the lab. Oyster husbandry followed methods of Bible and Sanford (2016) Local adaptation in an estuarine foundation species: Implications  for restoration. Biological Conservation. 193: 95-102.

extract_protocol:
Following salinity treatments, oysters were wrapped in aluminum foil, and flash frozen in liquid nitrogen. Whole frozen oysters were dissected from shells and transferred to 2.0 mL microcentrifuge tube containing 1 mL of Trizol® (Life Technologies). Samples were homogenized in TRIzol® using a TissueLyser LT for 4 minutes at 50 Hz and by subsequently drawing and expelling the resulting homogenate though a 20-gauge (0.99mm) needle and syringe until no tissue debris was visible. RNA was extracted using the guanidine isothiocyanate method (Chomczynski & Sacchi 1987). Following extraction, RNA was processed to remove tRNA and degraded fragments using a RNeasy® Mini Kit according to manufacturer’s instructions (Qiagen Inc., Valencia, CA, USA). RNA yield and purity were assessed using NanoDrop spectrophotometry. Following manufacturer’s recommendations, only RNA samples with A260:A280 and A260:A230 ratios greater than 1.8 and 2.0, respectively, were used in downstream steps.
A total of 36 cDNA libraries were created for RNA sequencing, corresponding to six replicate oyster samples from each of three populations (LL, OP, M) across two salinity treatments (5, 34 ppt). Purified total RNA was used to create each library using NEB Next Ultra Directional RNA Library Prep Kit for the Illumina sequencing platform (New England Biolabs) according to manufacturer’s instructions. Library quality was assessed using Bioanalyzer traces (Agilent Technologies, Santa Clara, CA, USA).

treatment_protocol: F2 oysters were exposed to either low (5 ppt) or control (34 ppt) salinity treatments in a room held at 12–13 °C. Salinity within the 5 ppt treatment tanks was lowered from 34 ppt over the course of five days and remained at ~5 ppt for four days before oysters from this treatment were sampled. Oysters in the control treatment were maintained at ~34 ppt over the same nine day period. Salinities were maintained by pre-mixing seawater and distilled water before performing daily water changes. Equal amounts of Shellfish Diet were provided to each exposure tank daily.

tag: population

value: Marshall

tag: salinity treatment

value: 5 ppt salinity (low salinity)

value: whole adult oysters


title: Whole organism, Loch Lomond population, 33 ppt treatment, replicate 6

description:
Sample 6
TE34C_S47_L005_R1

source_name: Whole organism_Loch Lomond population_33 ppt treatment

value: Loch Lomond

value: 33 ppt salinity (control)


title: Whole organism, Marshall population, 5 ppt treatment, replicate 6

description:
Sample 36
TE35C_S48_L005_R1


title: Whole organism, Loch Lomond population, 5 ppt treatment, replicate 6

description:
Sample 24
TE33C_S46_L005_R1

source_name: Whole organism_Loch Lomond population_5 ppt treatment


title: Whole organism, Loch Lomond population, 5 ppt treatment, replicate 5

description:
Sample 23
TE25C_S38_L005_R1


title: Whole organism, Loch Lomond population, 33 ppt treatment, replicate 5

description:
Sample 5
TE28C_S41_L005_R1


title: Whole organism, Oyster Point population, 5 ppt treatment, replicate 4

description:
Sample 28
TE26C_S39_L005_R1

source_name: Whole organism_Oyster Point population_5 ppt treatment

value: Oyster Point


title: Whole organism, Oyster Point population, 5 ppt treatment, replicate 5

description:
Sample 29
TE27C_S40_L005_R1


title: Whole organism, Marshall population, 33 ppt treatment, replicate 6

description:
Sample 18
TE32C_S45_L005_R1

source_name: Whole organism_Marshall population_33 ppt treatment


title: Whole organism, Oyster Point population, 5 ppt treatment, replicate 6

description:
Sample 30
TE29C_S42_L005_R1


title: nr4

tag: smokingstatus

value: NEVER

tag: Sex

value: female

tag: age

tag: eversmoked

value: no

tag: neversmoked

value: yes

tag: yearssmoked

tag: height

tag: weight

tag: waist

tag: hip

tag: barcode


title: Whole organism, Marshall population, 33 ppt treatment, replicate 5

description:
Sample 17
TE30C_S43_L005_R1

tag: tissue


title: Whole organism, Loch Lomond population, 33 ppt treatment, replicate 3

description:
Sample 3
TE22B_S35_L004_R1


title: Whole organism, Marshall population, 33 ppt treatment, replicate 4

description:
Sample 16
TE24B_S37_L004_R1


title: Whole organism, Loch Lomond population, 33 ppt treatment, replicate 4

description:
Sample 4
TE23B_S36_L004_R1


title: Whole organism, Marshall population, 33 ppt treatment, replicate 3

description:
Sample 15
TE21B_S34_L004_R1


title: Whole organism, Loch Lomond population, 5 ppt treatment, replicate 4

description:
Sample 22
TE20B_S33_L004_R1


title: Whole organism, Marshall population, 33 ppt treatment, replicate 2

description:
Sample 14
TE18B_S31_L004_R1


title: Whole organism, Marshall population, 5 ppt treatment, replicate 4

description:
Sample 34
TE19B_S32_L004_R1


title: Whole organism, Oyster Point population, 33 ppt treatment, replicate 5

description:
Sample 11
TE17B_S30_L004_R1

source_name: Whole organism_Oyster Point population_33 ppt treatment


title: Whole organism, Oyster Point population, 33 ppt treatment, replicate 4

description:
Sample 10
TE16B_S29_L004_R1


title: Whole organism, Oyster Point population, 33 ppt treatment, replicate 3

description:
Sample 9
TE15B_S28_L004_R1


title: Whole organism, Oyster Point population, 33 ppt treatment, replicate 2

description:
Sample 8
TE14B_S27_L004_R1


title: Whole organism, Loch Lomond population, 5 ppt treatment, replicate 3

description:
Sample 21
TE13B_S26_L004_R1


title: Whole organism, Marshall population, 5 ppt treatment, replicate 3

description:
Sample 33
TE11A_S24_L003_R1


title: Whole organism, Loch Lomond population, 5 ppt treatment, replicate 2

description:
Sample 20
TE12A_S25_L003_R1


title: Whole organism, Oyster Point population, 33 ppt treatment, replicate 1

description:
Sample 7
TE10A_S23_L003_R1


title: Whole organism, Loch Lomond population, 33 ppt treatment, replicate 2

description:
Sample 2
TE09A_S22_L003_R1


title: Whole organism, Oyster Point population, 5 ppt treatment, replicate 3

description:
Sample 27
TE08A_S21_L003_R1


title: Whole organism, Oyster Point population, 5 ppt treatment, replicate 2

description:
Sample 26
TE07A_S20_L003_R1


title: Whole organism, Oyster Point population, 33 ppt treatment, replicate 6

description:
Sample 12
TE36C_S49_L005_R1


title: Whole organism, Loch Lomond population, 33 ppt treatment, replicate 1

description:
Sample 1
TE05A_S18_L003_R1


title: Whole organism, Marshall population, 33 ppt treatment, replicate 1

description:
Sample 13
TE01A_S14_L003_R1


title: Whole organism, Marshall population, 5 ppt treatment, replicate 1

description:
Sample 31
TE02A_S15_L003_R1


title: Whole organism, Oyster Point population, 5 ppt treatment, replicate 1

description:
Sample 25
TE03A_S16_L003_R1


title: GFP+ target cells from sort 1

city: Ashburn

first: Ella

last: Preger-Ben Noon

state: VA

address: 19700 Helix Drive

institute: HHMI Janelia Research Campus

description:
Sample_EP2
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz

data_processing:
To remove artifacts resulting from amplification of RNA with the Nugen Ovation Kit we trimmed five nucleotides from the 5' end of RNA-seq reads using FASTX (http://hannonlab.cshl.edu/fastx_toolkit).
Trimmed reads were aligned to the D. melanogaster genome (version dm3) and to the ERCC spike-in sequences using TOPHAT v1.4.0 (Trapnell et al., 2009) with options "--no-novel-juncs --min-intron-length 30 --max-intron-length 200000" and guided by FlyBase gene annotations ( r5.41; Attrill et al., 2015).
We estimated transcript and gene expression levels using CUFFLINKS v1.3.0 (Trapnell et al., 2010) with options for multi-hit read correction, fragment bias correction, and masking of mitochondrial and noncoding transcripts.
We identified differentially expressed genes between pairs of samples at a false discovery rate of 5% using CUFFDIFF v1.3.0
Genome_build: UCSC dm3
Supplementary_files_format_and_content: [genes_rpkm_table.txt.gz] a tab-delimited text file of gene-level CUFFLINK abundance estimates and CUFFDIFF differential expression significance
Supplementary_files_format_and_content: [isoforms_rpkm_table.txt.gz] a tab-delimited text file of isoform-level CUFFLINK abundance estimates and CUFFDIFF differential expression significance

organism: Drosophila melanogaster

source_name: GFP+ target cells from sort, embryo

growth_protocol: Approximately 50 ml of young wild type flies or flies carrying the E10::EGFP transgene were reared in large population cages at 25°C. Following one hour of egg collection, embryos were left to age for approximately eleven hours at 25°C.

extract_protocol:
RNA was purify using Qiagen RNeasy Plus Micro kit according to the manufacturer's instructions
Five ng of total RNA was used for cDNA synthesis and amplification using the Ovation RNA-Seq System V2 (NuGen). To quantify the RNA-seq results, we added ERCC RNA Spike-In Control Mix 2 (1 µl of 1:10000 dilution, Ambion) to the samples prior to amplification. One µg of each amplified sample was used for library construction using the Encore NGS Library System I (NuGen)

treatment_protocol: Cells from early stage 14 embryos were dissociated using the method described in Salmand et. al. (Salmand et al., 2011) and EGFP-positive cells were sorted on a Becton-Dickinson FACSVantage SE w/Diva flow cytometer equipped with a 488 nm argon laser as follows: Gating parameters were set on cells isolated from wild type embryos and then used to sort EGFP-positive cells from E10::EGFP embryos. Cells were first discriminated from all events by analyzing the Forword scatter (FSC)/ Side Scatter (SSC) ratio (Cells). Then, single cells were selected using SSC and FSC parameters (Figure S4A). On the FSC population, the GFP positive cells were identified based on fluorescence intensity in the GFP channel versus autofluorescence at 575 nm. EGFP-positive cells were sorted through a flow chamber with a 70 μm flow cell tip under 15 psi sheath fluid pressure and collected in RNase-free PBS. At the end of the sorting, 50,000 cells from the parental FSC population were collected and used to represent all embryonic cells. The collected cells were centrifuged at 850 g for 10 minutes at 4°C, resuspended in 350 µl RTL buffer (Qiagen),

tag: developmental stage

value: early stage 14

tag: cell type

value: E10::EGFP positive


title: FSC all cells from sort 1

description:
Sample_EP1
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz

source_name: FSC all cells from sort, embryo

value: all embryonic cells


title: FSC all cells from sort 2

description:
Sample_EP3
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz


title: GFP+ target cells from sort 3

description:
Sample_EP6
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz


title: GFP+ target cells from sort 4

description:
Sample_EP8
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz


title: GFP+ target cells from sort 2

description:
Sample_EP4
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz


title: wt_p4_S8

status: Public on Apr 27 2018

city: Bethesda

first: Jeffrey

middle: Allen

last: Farrell

state: MD

address: 6 Center Drive (6A/1A06)

department: Division of Developmental Biology

institute: National Institute of Child Health and Human Development

description:
Full-length fragments
SS2_fpkm.txt.gz

data_processing:
Bases 1-8 (the UMI and template switch) were trimmed from the remaining reads, trimmed stretches of polyA or polyT longer than 6bp and removed any reads that were shorter than 15 bp after trimming.
We identified and depleted mtRNA and rRNA reads from the remaining reads by using Bowtie2 to align all reads against a transcriptome of mtRNA and rRNA, using the parameters -q --phred33 -N 1 -I 1 -X 2000 -k 200 --score-min L,3.36,-0.617 --no-discordant --no-mixed --no-unal.
Remaining reads were then aligned to a modified version of the Ensembl Zv10 release 81 zebrafish transcriptome using Bowtie2 with parameters -q --phred33 -N 1 -I 1 -X 2000 -k 200 --score-min L,3.36,-0.617 --no-discordant --no-mixed. The transcriptome start site of each gene was padded by 100bp to increase alignment rates, since reads were generated from the 5’ end of the transcript, and not all transcripts had correct annotations for their 5’ end. Additionally, pseudogenes were removed from the transcriptome (as they sometimes interfered with real genes, given our 5’ coordinate selection described below).
We chose the best alignment per read as the read with the most 5’ alignment coordinate to a transcript among those that aligned to the correct strand and had an alignment score of no more than 4 below the best alignment score overall. We then iteratively collapsed pairs of UMIs, if the sum of the minimum Phred quality scores across non-matching bases was at most 30 (to account for mis-matched bases that were likely due to sequencing errors). A collapsed pair was represented for future comparisons by the UMI with a larger prior number of aligned reads unless the other UMI had at least 75% as many reads aligned and had equal or better UMI quality scores for those alignments (evaluated as the softmin over aligned reads of the softmax over quality scores for the UMI bases in an aligned read).
After collapsing, the number of transcripts observed per gene was calculated. This value was then adjusted based on the probability of a collision (the drawing at random of the same UMI multiple times), which was calculated from the number of UMIs not detected for a given gene.
The full length fragmented reads without 5’ enrichment or UMI were aligned using RSEM against a reference transcriptome, which is depleted of mtRNA, rRNA, and pseudogenes as described above. The command ‘rsem-calculate-expression’ was used with parameters -p 4 --paired-end --seed-length 24 for performing the alignment. The resulting gene levels in FPKM was used to build the expression matrix.
Genome_build: GRCz10 dr81
Supplementary_files_format_and_content: Tab-delimited text files include gene levels (UMI counts or FKPM) for each sample.

organism: Danio rerio

source_name: Embryonic cell

growth_protocol: Fish of each genotype were incrossed and embryos were collected. Embryos were dechorionated and allowed to grow at 28°C in standard fish water containing methylene blue till 50% epiboly stage.

extract_protocol:
50% epiboly embryos were manually deyolked and mechanically dissociated. Single cells were manually picked under a dissecting microscope and flash frozen on dry ice in 4uL lysis buffer.
Libraries were prepared using a SMART-seq2 protocol (Picelli et al., 2014) with custom TSO oligos and indexing adapters (Satija et al., 2015). Reaction volumn for tagmentation steps were reduced to 15% of the volumn indicated in the protocol. 88 MZoep libraries were built with customized indices for 5’-enriched transcripts with unique molecular identifiers (UMIs). The remaining libraries were built without UMIs, and contained fragments along full-length transcripts.

value: 50% epiboly

tag: library preparation batch

tag: genotype

value: Wild-type TLAB


title: wt_p4_S9

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S6

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S7

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S5

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S3

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S4

description:
Full-length fragments
SS2_fpkm.txt.gz


title: FSC all cells from sort 4

description:
Sample_EP7
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz


title: wt_p4_S12

description:
Full-length fragments
SS2_fpkm.txt.gz


title: Whole organism, Loch Lomond population, 5 ppt treatment, replicate 1

description:
Sample 19
TE06A_S19_L003_R1

tag: population

tag: salinity treatment


title: Whole organism, Marshall population, 5 ppt treatment, replicate 2

description:
Sample 32
TE04A_S17_L003_R1


title: wt_p4_S13

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S10

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S2

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S9

description:
Full-length fragments
SS2_fpkm.txt.gz


title: FSC all cells from sort 3

description:
Sample_EP5
genes_rpkm_table.txt.gz
isoforms_rpkm_table.txt.gz


title: wt_p3_S8

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S5

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S7

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S4

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S3

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S2

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S1

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S20

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S19

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S1

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S16

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S21

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S22

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S18

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S15

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S14

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S13

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S12

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S11

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S10

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S9

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S8

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S6

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S6

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S7

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p3_S17

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S3

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S11

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S4

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S14

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S13

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S12

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S10

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p1_S3

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p1_S1

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p1_S2

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S5

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p2_S1

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S3

description:
Full-length fragments
SS2_fpkm.txt.gz

value: MZoep tz57


title: oep_p4_S2

description:
Full-length fragments
SS2_fpkm.txt.gz


title: wt_p4_S11

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S8

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S24

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S27

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S23

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S7

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S5

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S25

description:
Full-length fragments
SS2_fpkm.txt.gz


title: oep_p4_S21

description:
Full-length fragments
SS2_fpkm.txt.gz


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Full-length fragments
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