title: LT_old_mouse3_plate1_stim_sc_G1_S169


title: LT_old_mouse3_plate1_stim_sc_H3_S183

status: Public on Jul 19 2017

type: SRA

city: Cambridge

first: Carl

middle: G

last: de Boer

state: MA

address: 415 Main St

country: USA

institute: The Broad Institute

library_source: transcriptomic

data_processing:
Mapping reads to transcriptome with RSEM
Flag cells due to: not a single cell (100 cells; pop); low percent of reads mapping to transcriptome (pctTrans); low number of genes detected (fewGenes); cells undergoing cell cycle (inferred from expression signature; cycling); contaminating plasma cell, based on gene expression signature (plasma)
Genome_build: mm9
Supplementary_files_format_and_content: TPM and raw counts for all single cell samples

molecule: polyA RNA

organism: Mus musculus

source_name: HSCs identified by cell surface markers, isolated from mouse bone marrow.

growth_protocol: All cells were grown at 37C and 5%CO2 in HSPC media.

extract_protocol:
Single cell and 100 cell populations were lysed by sorting into 5ul TCL Buffer (QIAGEN) with 1% 2-Mercaptoethanol, centrifuged and frozen at -80°C - this was used for SMART-seq2 as described in Picelli et al. Nat. Prot. 2014.
SMART-seq2

treatment_protocol: Where applicable, treated HSCs were grown in HSPC media with 100ng/ml LPS (SIGMA CAT: L2880-100MG), and 1ug/ml Pam3csk4 (Invivogen CAT: 41-11-60-10) for two hours

tag: cell type

value: LT

tag: mouse age

value: old

tag: mouse id

tag: treatment

value: LPS+PAM stimulation

tag: plate id

tag: well id

tag: sample type

value: single cell

tag: qc flags


title: LT_old_mouse3_plate1_stim_sc_D3_S135


title: LT_old_mouse3_plate1_stim_sc_E1_S145


title: LT_old_mouse3_plate1_stim_sc_E2_S146


title: LT_old_mouse3_plate1_stim_sc_F4_S160


title: LT_old_mouse3_plate1_stim_sc_E3_S147


title: LT_old_mouse3_plate1_stim_sc_D2_S134


title: LT_old_mouse3_plate1_stim_sc_D1_S133


title: LT_old_mouse3_plate1_stim_sc_B2_S110

value: cycling


title: LT_old_mouse3_plate1_stim_sc_C3_S123


title: LT_old_mouse3_plate1_stim_sc_H1_S181


title: LT_old_mouse3_plate1_stim_sc_C2_S122


title: LT_old_mouse3_plate1_stim_sc_C1_S121


title: LT_old_mouse3_plate1_stim_sc_C4_S124


title: LT_old_mouse3_plate1_stim_sc_B3_S111


title: LT_old_mouse3_plate1_stim_sc_B1_S109


title: LT_old_mouse3_plate1_stim_sc_D4_S136


title: LT_old_mouse3_plate1_stim_pop_H4_S184

value: 100 cell population


title: LT_old_mouse3_plate1_stim_sc_A3_S99


title: LT_old_mouse3_plate1_stim_sc_A1_S97


title: LT_old_mouse3_plate1_nostim_sc_H2_S86

value: no stimulation


title: LT_old_mouse3_plate1_stim_sc_A2_S98


title: LT_old_mouse3_plate1_nostim_sc_H3_S87


title: LT_old_mouse3_plate1_nostim_sc_H1_S85


title: LT_old_mouse3_plate1_nostim_sc_G2_S74


title: LT_old_mouse3_plate1_nostim_sc_F4_S64


title: LT_old_mouse3_plate1_nostim_sc_G1_S73


title: LT_old_mouse3_plate1_nostim_sc_G4_S76


title: LT_old_mouse3_plate1_nostim_sc_F3_S63


title: vector_RNA-Seq

city: wuhan

first: xiaohua

last: chen

address: donghu road 185

department: medical genetics

country: China

institute: wuhan university

data_processing:
Sequencing reads generated were mapped to human genome (hg38) database using Burrows-Wheeler Aligner
Peak calling was performed by MACS 2.0 and significant enrichments were identified with threshold level set at q-value <0.05.
Purity of RNA samples was determined with NanoPhotometer spectrophotometer (Implen, CA, USA) and concentration was quantified in a Qubit 2.0 fluorometer (Life Technologies).
Sequencing libraries were constructed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, E7420) and library quality was validated on Bioanalyzer 2100 (Agilent Technologies).
Clustered sequences were generated using TruSeq SE Cluster Kit v3-cBot-HS (Illumina) followed by sequencing on HiSeq 2500 (Illumina).
Genome_build: hg38
Supplementary_files_format_and_content: bigwig and FPKM values

molecule: total RNA

organism: Homo sapiens

source_name: cervical cancer cells

extract_protocol:
Clarified lysates were diluted with ChIP dilution buffer,precleared with salmon sperm DNA blocked Protein G Sepharose 4 Fast Flow   for 1 h at 4°c and incubated overnight at 4°c with primary antibodies respectively. Protein-DNA complex is then precipitated with salmon sperm DNA blocked Protein G Sepharose beads at 4°c for 4 h. Bound complexes were washed once .ChIP DNA were prepared from the overnight samples. Total RNA from  cells were extracted using TRIzol reagent following the manufacturer's instructions.
Sequencing of samples was performed on HiSeq 2500 (Illumina, CA, USA) platform according to Illumina’s protocol.Directional RNA Library Prep Kit for Illumina® (NEB, USA)

tag: genotype/variation

value: vector


title: KDM5C_RNA-Seq

value: KDM5C overexpression


title: vector_Input_ChIPSeq

library_source: genomic

molecule: genomic DNA


title: vector_H3K27Ac_ChIPSeq

tag: chip antibody

value: H3K27Ac(Cell Signaling Technology, 8173)


title: LT_old_mouse3_plate1_stim_sc_B4_S112


title: LT_old_mouse3_plate1_nostim_sc_F2_S62


title: LT_old_mouse3_plate1_stim_sc_A4_S100


title: LT_old_mouse3_plate1_nostim_sc_F1_S61


title: vector_H3K4me3_ChIPSeq

value: H3K4me3(ABclonal, A2357)


title: LT_old_mouse3_plate1_nostim_sc_E4_S52


title: parental_Input_ChIPSeq

value: wild type


title: vector_H3K4me1_ChIPSeq

value: H3K4me1(ABclonal, A2355)


title: LT_old_mouse3_plate1_nostim_sc_E3_S51


title: parental_H3K27Ac_ChIPSeq


title: LT_old_mouse3_plate1_nostim_sc_G3_S75


title: KDM5C_H3K27Ac_ChIPSeq


title: LT_old_mouse3_plate1_nostim_sc_E2_S50


title: LT_old_mouse3_plate1_nostim_sc_D4_S40


title: KDM5C_Input_ChIPSeq


title: KDM5C_H3K4me3_ChIPSeq


title: KDM5C_H3K4me1_ChIPSeq


title: LT_old_mouse3_plate1_nostim_sc_D3_S39


title: LT_old_mouse3_plate1_nostim_sc_D2_S38


title: LT_old_mouse3_plate1_nostim_sc_D1_S37


title: LT_old_mouse3_plate1_nostim_sc_C3_S27


title: LT_old_mouse3_plate1_nostim_sc_C4_S28


title: LT_old_mouse3_plate1_nostim_sc_C1_S25


title: IP for eH3.3 atrx-1 rep1

city: Aubière

first: Céline

last: Duc

address: 10 Avenue Blaise Pascal

country: France

institute: CNRS

description: processed data file: d2.Fnor.smooth.wig

data_processing:
Quality check on reads was done using FASTQC with a cut-off Phred score of 20.
Reads with minimum length of 36 bp was kept after trimming using Trimmomatic (minimum length of 36 bp; mean Phred quality score greater than 30; leading and trailing bases removal with base quality below 3; sliding window of 4:15).
ChIP-Seq reads were aligned onto TAIR10 using Bowtie  with unique read mapping parameter.
Peak detection was performed using MACS2 and changes in location and occupancy of eH3.3 binding position were performed with Danpos2 (minimum read depth:5, window size:50bp, minimal distance between peaks:100bp). Mean and standard deviation of the coverage depth were calculated and plotted using Qualimap.
Genome_build: TAIR10
Supplementary_files_format_and_content: For d1.Fnor.smooth.wig and d2.Fnor.smooth.wig, peaks for combined replicates of each genotype were displayed. The d1-d2.positions.integrative.xls file displays results of the comparison of both genotypes using DANPOS2 to quantify changes in position, summit and point occupancy as well as fuzziness.

organism: Arabidopsis thaliana

source_name: atrx-1 mutant_IP for eH3.3 atrx-1

growth_protocol: 2.5-week-old in vitro grown plantlets

extract_protocol: ChIP was performed according to Veluchamy et al., 2016  [PMID: 27410265] with Anti-FLAG® M2 Magnetic beads (Sigma)

tag: ecotype background

value: col

value: atrx-1 mutant

tag: age

tag: tissue

value: in vitro grown plantlets

value: Anti-FLAG® M2 Magnetic beads (Sigma)


title: IP for eH3.3 rep2

description: processed data file: d1.Fnor.smooth.wig

source_name: wild type_IP for eH3.3


title: LT_old_mouse3_plate1_nostim_sc_C2_S26

value: LT


title: IP for eH3.3 rep1

description: processed data file: d1.Fnor.smooth.wig


title: LT_old_mouse3_plate1_nostim_sc_B2_S14

value: old


title: input DNA

city: daejeon

first: wonyul

last: jang

state: State...

address: yuseong-gu, daehakro

country: South Korea

institute: KAIST (Korea Advanced Institute of Science and Technology)

data_processing:
Sequences: Standard Illumina software base-calling and quality-control filtering was applied
Sequences (50 nt reads, single end) were aligned to the human genome (hg19) using the BWA algorithm (v0.6.1, default parameters).
Aligns were extended in silico at their 3’-ends to a length of 150 bp, which is the average genomic fragment length in the size-selected library, and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWIG files.
Peak locations were determined using the MACS algorithm (v1.4.2) with a cutoff of pvalue = 1E-10
Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations
Genome_build: hg19
Supplementary_files_format_and_content: BW = signal map in bigWIG format (UCSC)
Supplementary_files_format_and_content: BED = standard BED file containing MACS peaks

source_name: MCF10A non-transformed mammary cells

growth_protocol: Cells were growin in DMEM/F12 supplemented with 5% horse serum (Invitrogen), 20 ng/ml EGF (Peprotech), 10 mg/ml insulin (Sigma), 0.5 mg/ml hydrocortisone (Sigma), 100 ng/ml cholera toxin (Sigma) and penicillin/streptomycin (Invitrogen).

extract_protocol:
Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine.  Lysates were sonicated with a microtip sonicator to shear the DNA. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Starting with 50 ug of chromatin, genomic DNA regions of interest were isolated using Flag-M2 affinity beads. Immune complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on HiSeq 2500.

tag: cell line

value: breast cancer cell line

value: none (input)


title: Input for eH3.3

source_name: wild type_Input for eH3.3


title: LT_old_mouse3_plate1_nostim_sc_B1_S13

value: single cell


title: LT_old_mouse3_plate1_nostim_sc_B4_S16


title: LT_old_mouse3_plate1_nostim_sc_A3_S3


title: LT_old_mouse3_plate1_nostim_sc_B3_S15


title: LT_old_mouse3_plate1_nostim_sc_A4_S4


title: LT_old_mouse3_plate1_nostim_sc_A1_S1


title: LT_old_mouse2_plate1_stim_sc_H3_S375

value: LPS+PAM stimulation

value: cycling


title: LT_old_mouse2_plate1_stim_sc_G2_S362


title: LT_old_mouse2_plate1_stim_sc_H1_S373


title: LT_old_mouse2_plate1_stim_sc_G4_S364


title: LT_old_mouse2_plate1_stim_sc_H2_S374


title: LT_old_mouse3_plate1_nostim_sc_E1_S49

value: no stimulation


title: LT_old_mouse3_plate1_nostim_sc_A2_S2


title: LT_old_mouse2_plate1_stim_sc_G3_S363


title: LT_old_mouse2_plate1_stim_sc_G1_S361


title: Flag-YAP-5SA_ChIPSeq

value: Flag-M2 (Sigma, A2220)


title: LT_old_mouse3_plate1_nostim_pop_H4_S88

value: 100 cell population

value: pop


title: IP for eH3.3 atrx-1 rep2

description: processed data file: d2.Fnor.smooth.wig


title: LT_old_mouse2_plate1_stim_sc_F4_S352

value: fewGenes


title: LT_old_mouse2_plate1_stim_sc_D2_S326


title: LT_old_mouse2_plate1_stim_sc_F2_S350

value: pctTrans


title: LT_old_mouse2_plate1_stim_sc_D4_S328


title: LT_old_mouse2_plate1_stim_sc_C3_S315


title: LT_old_mouse2_plate1_stim_sc_C4_S316


title: LT_old_mouse2_plate1_stim_sc_F1_S349


title: LT_old_mouse2_plate1_stim_sc_F3_S351


title: LT_old_mouse2_plate1_stim_sc_C2_S314


title: LT_old_mouse2_plate1_stim_sc_E1_S337


title: LT_old_mouse2_plate1_stim_sc_A2_S290


title: LT_old_mouse2_plate1_stim_sc_B4_S304


title: LT_old_mouse2_plate1_stim_sc_D3_S327


title: LT_old_mouse2_plate1_nostim_sc_H1_S277


title: LT_old_mouse2_plate1_stim_sc_E2_S338


title: LT_old_mouse2_plate1_stim_sc_B2_S302


title: LT_old_mouse2_plate1_stim_sc_E4_S340


title: LT_old_mouse2_plate1_stim_sc_E3_S339


title: LT_old_mouse2_plate1_nostim_sc_H3_S279


title: LT_old_mouse2_plate1_stim_sc_D1_S325


title: KLF4 ChIP KLF4-3F Rep2

city: Paris

first: Mickael

last: Orgeur

address: 25-28 rue du Docteur Roux

institute: Institut Pasteur

data_processing:
ChIP-seq single-end reads were filtered on their quality by using the FASTX-Toolkit (median quality value of minimum 28).
Filtered reads were mapped against the chicken genome galGal4 by using BWA (default parameters).
Uniquely mapped reads were extracted and duplicated reads were removed by using the tool rmdup from SAMtools.
Peak calling was performed as suggested by the ENCODE consortium by using MACS2 (--bw 130/135; -g 1.0e9; --to-large; -p 0.025).
Significant peaks were selected based on an IDR analysis (peak.half.width -1; min.overlap.ratio 0; is.broadpeak F; ranking.measure p.value) with a threshold fixed at 0.01.
Genome_build: galGal4
Supplementary_files_format_and_content: Peaks called by MACS2 in ENCODE narrowPeak format.

organism: Gallus gallus

source_name: mesenchymal cells

growth_protocol: Mesenchymal cells were isolated from limb buds of E4.5 chick embryos and were plated as 10-µL droplets at a concentration of 2.10^7 cells/mL. Cells were maintained in culture for 5 days at 37°C in DMEM/Ham’s F-12 (1:1) medium (Biochrom) supplemented with 10% FBS (Biochrom), 0.2% chicken serum (Sigma-Aldrich), 1% L-glutamine (Lonza) and 1% penicillin/streptomycin (Lonza).

extract_protocol:
Mesenchymal cells harvested from 24 cultures were cross-linked in 1% FA for 10 min on ice. Following cell lysis and nuclei isolation, nuclear extracts were sonicated to fragment chromatin between 200 and 500 bp. 30 µg of chromatin extracts were mixed with 10 µg of anti-FLAG (Sigma-Aldrich, F1804). Antibody-TF-DNA complexes were pulled down by using 40 µL of magnetic beads (Dynabeads protein G; Thermo Fischer). Enriched complexes were eluted from the beads. Reverse cross-linking was performed overnight at 65°C. DNA was purified by successive treatments with RNase A and Proteinase K followed by ethanol precipitation.
ChIP and input librairies were prepared by using the NEBNext Ultra DNA Library Preparation kit for Illumina (New England Biolabs). 50-bp single-end reads were generated by using a HiSeq 1500 sequencer (Illumina).

treatment_protocol: Prior to seeding, mesenchymal cells were infected with RCAS-BP(A) retroviral particles carrying the CDS of connective tissue-asociated transcription factors.

tag: breed

value: Lohmann Selected Leghorn

value: limb buds

value: mesenchymal cells

tag: developmental stage

tag: time of culture

value: 5 days

tag: condition

tag: antibody

value: mouse anti-FLAG (Sigma-Aldrich, F1804)


title: LT_old_mouse2_plate1_stim_sc_B3_S303

tag: cell type

value: LT

tag: mouse age

value: old

tag: mouse id


title: LT_old_mouse2_plate1_stim_sc_B1_S301

tag: treatment

tag: plate id

tag: well id


title: LT_old_mouse2_plate1_stim_sc_A4_S292

tag: sample type


title: LT_old_mouse2_plate1_nostim_sc_G4_S268


title: LT_old_mouse2_plate1_nostim_sc_H2_S278


title: LT_old_mouse2_plate1_stim_sc_A1_S289


title: KLF4 ChIP Input Rep2


title: LT_old_mouse2_plate1_stim_sc_A3_S291


title: LT_old_mouse2_plate1_nostim_sc_G3_S267


title: LT_old_mouse2_plate1_stim_pop_H4_S376

tag: qc flags


title: KLF4 ChIP KLF4-3F Rep1


title: KLF4 ChIP Input Rep1


title: LT_old_mouse2_plate1_nostim_sc_G2_S266


title: KLF2 ChIP KLF2-3F Rep2


title: KLF2 ChIP Input Rep2


title: KLF2 ChIP Input Rep1


title: LT_old_mouse2_plate1_nostim_sc_F2_S254


title: LT_old_mouse2_plate1_nostim_sc_F3_S255


title: LT_old_mouse2_plate1_nostim_sc_F4_S256


title: EGR1 ChIP EGR1-3F Rep2


title: LT_old_mouse2_plate1_nostim_sc_F1_S253


title: LT_old_mouse2_plate1_nostim_sc_E4_S244


title: KLF2 ChIP KLF2-3F Rep1


title: LT_old_mouse2_plate1_nostim_sc_E3_S243


title: LT_old_mouse2_plate1_nostim_sc_E2_S242


title: LT_old_mouse2_plate1_nostim_sc_G1_S265


title: EGR1 ChIP EGR1-3F Rep1


title: LT_old_mouse2_plate1_nostim_sc_E1_S241


title: LT_old_mouse2_plate1_nostim_sc_D4_S232


title: OSR2 ChIP OSR2-3F Rep2


title: EGR1 ChIP Input Rep1


title: OSR2 ChIP Input Rep2


title: LT_old_mouse2_plate1_nostim_sc_D3_S231


title: OSR2 ChIP OSR2-3F Rep1


title: LT_old_mouse2_plate1_nostim_sc_D2_S230


title: OSR2 ChIP Input Rep1


title: LT_old_mouse2_plate1_nostim_sc_D1_S229


title: OSR1 ChIP OSR1-3F Rep2


title: LT_old_mouse2_plate1_nostim_sc_C3_S219


title: LT_old_mouse2_plate1_nostim_sc_C2_S218


title: OSR1 ChIP OSR1-3F Rep1


title: OSR1 ChIP Input Rep1


title: EGR1 ChIP Input Rep2


title: Control ChIP H3K27me3 Rep2

data_processing:
ChIP-seq single-end reads were filtered on their quality by using the FASTX-Toolkit (median quality value of minimum 28).
Filtered reads were mapped against the chicken genome galGal4 by using BWA (default parameters).
Uniquely mapped reads were extracted and duplicated reads were removed by using the tool rmdup from SAMtools.
Peak calling was performed as suggested by the ENCODE consortium and the Roadmap Epigenomics project by using MACS2 (--bw 400; -g 1.0e9; --to-large). Narrow peaks were called by using a p-value (-p) of 0.01. Broad peaks were called by using an additional broad-peak p-value (-p 0.01; --broad; --broad-cutoff) of 0.1.
Broad peaks that contain at least one narrow peak were extracted by using BEDtools intersect.
Genome_build: galGal4
Supplementary_files_format_and_content: Broad peaks called by MACS2 in ENCODE broadPeak format; Narrow peaks called by MACS2 in ENCODE narrowPeak format.

extract_protocol:
Mesenchymal cells harvested from 8 cultures were cross-linked in 1% FA for 10 min on ice. Following cell lysis and nuclei isolation, nuclear extracts were sonicated to fragment chromatin between 200 and 500 bp. 10 µg of chromatin extracts were mixed with 4 µg/4 µL of corresponding antibody. Antibody-histone-DNA complexes were pulled down by using 40 µL of magnetic beads (Dynabeads protein G; Thermo Fischer). Enriched complexes were eluted from the beads. Reverse cross-linking was performed overnight at 65°C. DNA was purified by successive treatments with RNase A and Proteinase K followed by ethanol precipitation.
ChIP and input librairies were prepared by using the NEBNext Ultra DNA Library Preparation kit for Illumina (New England Biolabs). 50-bp single-end reads were generated by using a HiSeq 1500 sequencer (Illumina).

treatment_protocol: Prior to seeding, mesenchymal cells were infected with empty RCAS-BP(A) retroviral particles carrying no recombinant protein.

value: RCAS-BP(A)-empty

value: mouse anti-H3K27me3 (Millipore, 07-449)


title: LT_old_mouse2_plate1_nostim_sc_B4_S208

value: single cell


title: LT_old_mouse2_plate1_nostim_sc_C1_S217


title: Control ChIP H3K27me3 Rep1


title: Control ChIP H3K27ac Rep2

value: mouse anti-H3K27ac (Abcam, ab4729)


title: LT_old_mouse2_plate1_nostim_sc_B3_S207

value: no stimulation


title: Control ChIP H3K4me3 Rep2

value: mouse anti-H3K4me3 (Millipore, 07-473)


title: LT_old_mouse2_plate1_nostim_sc_A4_S196


title: Control ChIP H3K27ac Rep1


title: LT_old_mouse2_plate1_nostim_sc_B1_S205


title: LT_old_mouse2_plate1_nostim_sc_B2_S206


title: Control ChIP H3K4me3 Rep1


title: LT_old_mouse2_plate1_nostim_sc_A3_S195


title: Control ChIP H3K4me2 Rep2

value: mouse anti-H3K4me2 (Abcam, ab32356)


title: LT_old_mouse2_plate1_nostim_sc_A2_S194


title: Control ChIP H3K4me2 Rep1


title: LT_old_mouse2_plate1_nostim_sc_A1_S193


title: LT_old_mouse2_plate1_nostim_pop_H4_S280

value: 100 cell population


title: Control ChIP H3K4me1 Rep2

value: mouse anti-H3K4me1 (Abcam, ab8895)


title: OSR1 ChIP Input Rep2


title: LT_old_mouse2_plate1_nostim_sc_C4_S220


title: miR-145 overexpressing cells

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: University of Groningen

description: RNA-seq on miR-145 overexpressing cells

data_processing:
5 bases were trimmed from reads' 5'-end, using FastX toolkit. Reads were aligned to the mm9 reference genome using TopHat with parameters "--bowtie1 -a 5". BedGraph track files were generated using the Wiggles utility shipped with the TopHat package.
Genome_build: mm9
Supplementary_files_format_and_content: BEDGRAPH

source_name: NMuMG

growth_protocol: NMuMG cells, were maintained in Dulbecco's modified Eagle's medium with 4.5 g/L glucose, supplemented with 10 mcg/ml insulin and 10% fetal bovine serum at 37°C, in a humidified atmosphere of 5% CO2 in air.

extract_protocol: Cells were washed twice in PBS, then 1 ml of ice-cold TRIzol was added directly to cells. Cells were scraped using a P1000 pipette, and solution was pipetted up-and-down to homogeneity. RNA extraction was performed following manufacturer instructions.

treatment_protocol: All stable cell lines were generated via lentiviral infection. Lentiviral particles were produced according to Trono’s lab protocol (http://tronolab.epfl.ch). Supernatant was harvested 48h post-transfection, filtered with 0.45 mm filters, diluted and used to transduce cells, in the presence of Polybrene (Sigma Aldrich, Saint Louis, MI, USA).

tag: genotype/variation

value: miR-145 overexpression


title: Control ChIP H3K4me1 Rep1

value: Lohmann Selected Leghorn


title: Control ChIP Input3 Rep2

value: limb buds


title: miR-143 overexpressing cells

description: RNA-seq on miR-143 overexpressing cells

value: miR-143 overexpression


title: Control replicate #2

description: RNA-seq on Control (untreated) cells

value: Control


title: Control ChIP Input1 Rep2

value: mesenchymal cells

value: 5 days


title: Control ChIP Input2 Rep2


title: Control ChIP Input3 Rep1


title: KLF4 RNA Rep1

data_processing:
RNA-seq strand-specific read pairs were mapped against the chicken genome galGal4 by using TopHat2 (-r 150; -N 3; --read-edit-dist 3; --library-type fr-firststrand; -i 50; -G) and a custom gene annotation model.
Alignment maps were split by strand by using SAMtools according to their FLAG field (strand plus: -f 128 -F 16, -f 80; strand minus: -f 144, -f 64 -F 16).
Fragments mapped on gene features were counted by using featureCounts (-p; -s 2; --ignoreDup; -B; -R).
Sample normalization and differential expression analysis was performed by using DESeq2 with a false-discovery rate (FDR; alpha) of 0.01.
Genome_build: galGal4
Supplementary_files_format_and_content: Tab-delimited text files containing the raw read counts for each gene for each replicate determined by featurecounts and used as input for DESeq2

extract_protocol:
RNA extracts were obtained by harvesting 6 cultures with RLT buffer (Qiagen). Total RNAs were purified by using the RNeasy mini kit (Qiagen) in combination to a DNase I (Qiagen) treatment to prevent genomic DNA contamination.
RNA libraries were prepared by using the TruSeq Stranded mRNA Library Preparation kit (Illumina). Strand-specific 50-bp paired-end reads were generated by using a HiSeq 2500 sequencer (Illumina) with a mean insert size of 150 bp.


title: Control replicate #1

description: RNA-seq on Control (untreated) cells


title: KLF4 RNA Rep2


title: d234_fb2.mRNAseq

city: Wageningen

first: Michael

middle: D

last: Nodine

state: Gelderland

address: Radix West

department: Plant Sciences

institute: Wageningen University & Research

description:
Paired-end 50 base read poly(A) RNA-Seq data from second biological replicate of dcl234 flowers
Processed data is Series supplementary file master_genes_Araport.txt

data_processing:
sRNA-Seq: Adaptors were removed, sequences were aligned to Col-0 genome (TAIR10) with Bowtie requiring perfect matches and allowing up to 100 alignments per read, and read values were normalized for the number of genome matches. For instance, if a read mapped perfectly to two locations, then 0.5 reads would be assigned to each of the two locations or loci.
sRNA-Seq: Reads were then assigned to mature miRNAs or tasiRNAs if they were 20-22 nt long and contained within +/- 2 nt of the sense strand of the miRNA or tasiRNA according to annotations in miRBase21 (Kozomara, A. & Griffiths-Jones, S. [2013] NAR) and Allen et al. (2005) Cell, respectively. Small RNA reads which were either 20-22 nt or 23-24 nt long and overlapped either strand of TAIR10 annotated transposons (i.e. transposable elements and transposable element genes) were assigned to the corresponding transposons they mapped to.
mRNA-Seq: Paired-end mRNA-Seq reads were aligned to the Arabidopsis thaliana Col-0 genome (TAIR10) and ERCC spike-ins using RSEM30 using default parameters. ERCC spike-in and Araport1131 transcript models were then quantified and recorded in "master_genes_Araport.txt".
Genome_build: TAIR10
Supplementary_files_format_and_content: *_miR_fams files contain sRNA-Seq RPM levels for miRNA families as annotated in miRBase21; *_tasiR_fams files contain sRNA-Seq RPM levels for tasiRNA families as annotated in Allen et al. (2005) Cell; *_siR_21 files contain sRNA-Seq RPM levels for 20-22 nt sRNAs for transposons as annotated in TAIR10; *_siR_24 files contain sRNA-Seq RPM levels for 23-24 nt sRNAs for transposons as annotated in TAIR10; master_genes_Araport.txt file contains mRNA-Seq levels for Araport11 annotated genes and ERCC spike-ins for all six samples (i.e. col0_leaf1, col0_leaf2, col0_fb1, col0_fb2, d234_fb1, d234_fb2).

source_name: unopened floral buds

growth_protocol: Controlled growth chamber at 20˚C-22˚C with a 16-h light/8-h dark cycle. Plants were 4-6 weeks old when tissue was harvested.

extract_protocol:
TRIzol reagent (Thermo Fisher Scientific)
For Samples 1 - 6, sRNA spike-in mix was added to total RNA followed by size-selection for 18-75 nt RNAs. The NEBnext small RNA library prep kit for Illumina (NEB) was then used to construct sRNA-Seq libraries. For Samples 7 - 12, ERCC spike-in mixes (LifeTech) were added to total RNA and 10 ng of total RNA/ERCC mix was used to generate mRNA-Seq libraries as described by Picelli et al. (2013) Nature Methods. All samples were sequenced on an Illumina Hi-Seq 2500 sequencing machine in either single read 50 base (sRNA-Seq) or paired-end read 50 base (mRNA-Seq) modes.

value: unopened floral buds

tag: ecotype

tag: genotype

value: triple homozygous mutant with dcl2-1, dcl3-1, dcl4-2t alleles


title: KLF2 RNA Rep1

tag: tissue


title: EGR1 RNA Rep2

tag: breed


title: KLF2 RNA Rep2

tag: developmental stage


title: Control ChIP Input2 Rep1

tag: time of culture

value: RCAS-BP(A)-empty


title: col0_fb1.mRNAseq

description:
Paired-end 50 base read poly(A) RNA-Seq data from first biological replicate of Col-0 flowers
Processed data is Series supplementary file master_genes_Araport.txt

value: wild type


title: d234_fb1.mRNAseq

description:
Paired-end 50 base read poly(A) RNA-Seq data from first biological replicate of dcl234 flowers
Processed data is Series supplementary file master_genes_Araport.txt


title: col0_fb2.mRNAseq

description:
Paired-end 50 base read poly(A) RNA-Seq data from second biological replicate of Col-0 flowers
Processed data is Series supplementary file master_genes_Araport.txt


title: EGR1 RNA Rep1

tag: condition


title: Clone #26 RRBS

first: Pierre-Antoine

last: Defossez

address: 35 rue Hélène Brion

department: UMR7216 "Epigenetics and Cell Fate"

institute: CNRS-Université Paris Diderot - Sorbonne Paris Cité

description: Bisulfited

data_processing:
Sequencing was realized by Integragen genomics on an Illumina HiSeq4000 platform. Image analysis and base calling is performed using Illumina Real Time Analysis ( 2.7.3 ) with default parameters.
Fastq files were first trimmed with trim_galore version 0.4.0 (--phred33 --length 40 --rrbs –non_directional --paired)
Fastq files were mapped to mouse genome (mm9) with bismark (bismark_v0.14.3)
Methylation state analysis has been performed with methylkit R package (version 0.9.5) and R-3.2.0.
CpG methylation was calculated over CpG with a minimum covarage of ten reads and a maximum coverage corresponding to the 99.9 percentil.
Genome_build: NCBI Build 37 – mm9
Supplementary_files_format_and_content: Bedgraph for methylation percentage were generated with methylkit

growth_protocol: Embryonic stem cells were cultured under standard feeder-free conditions on gelatinized tissue culture dishes with media containing DMEM (Gibco, 31966021) supplemented with 100 µM 2-mercaptoethanol (Gibco, 31350010), 1 x non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco), 1% penicillin-streptomycin, 15% KnockOut Serum Remplacement (Gibco, 10828028) and 103 U/ml leukemia inhibitory factor (LIF, Millipore) at 37 °C under an atmosphere of 5% CO2.

extract_protocol:
Genomic DNA  with Qiagen All Prep DNA/RNA miniprep, Cat#80204
RRBS protocol as described in Gu et al., 2011 (Nature Protocols) doi:10.1038/nprot.2010.190

value: Lig1 CRISPR mutant clone#26

value: Embryonic stem cell

tag: strain


title: Clone #9 RRBS

description: Bisulfited

value: Lig1 CRISPR mutant clone#9

tag: cell type


title: col0_leaf1.mRNAseq

description:
Paired-end 50 base read poly(A) RNA-Seq data from first biological replicate of Col-0 leaves
Processed data is Series supplementary file master_genes_Araport.txt

source_name: rosette and cauline leaves

value: rosette and cauline leaves


title: OSR1 RNA Rep2


title: OSR2 RNA Rep1


title: E14 Np95-/- RRBS

description: Bisulfited


title: Clone #5 RRBS

description: Bisulfited

value: Lig1 CRISPR mutant clone#5


title: OSR1 RNA Rep1


title: d234_fb2.sRNAseq

description: Single-end 50 base read small RNA-Seq data from second biological replicate of dcl234 flowers


title: Control ChIP Input1 Rep1

tag: antibody


title: E14WT RRBS

description: Bisulfited


title: Control RNA Rep1


title: d234_fb1.sRNAseq

description: Single-end 50 base read small RNA-Seq data from first biological replicate of dcl234 flowers


title: Clone #32 RRBS

description: Bisulfited

value: Lig1 CRISPR mutant clone#32


title: Dextran Control 24H sample 2

city: St. Augustine

first: david

middle: k

last: Simmons

state: FL

address: 9505 Ocean Shore Blvd

department: Biology

institute: University of Florida

data_processing:
Read quality was checked with Fastqc  (https://www.bioinformatics.babraham.ac.uk/projects/) and the  first 12 bases were trimmed using the FASTX toolkit along with  the removal of any sequences with phred scores below 33
FastqMCF was used to remove any adaptor contamination  and homopolymers
The reads were mapped to the edited Nematostella reference transcriptome genomic filtered model set with the exclusion of mitochondrial  and ribosomal RNA sequences
Using Bowtie 2.2.0 beta3 with –verysensitive-local and -a flags Counts were generated using the custom script from (Helm RR, Siebert S, Tulin S, Smith J, Dunn CW (2013) Characterization of differential transcript abundance through time during Nematostella vectensis development. BMC  Genomics 14:266.)
Supplementary_files_format_and_content: Read counts before abundance measurement

organism: Nematostella vectensis

source_name: Animal Tissue total RNA

growth_protocol: The fertilized/microinjected eggs were cultured in filtered 1/3× seawater at 17 °C. Until RNA extraction

extract_protocol:
~1ug of total RNA was extracted from the samples using the Tri-reagent protocol
1ug of total RNA was enriched for Poly-A mRNA, and taken through library preparation for single-end 50 bp reads.

value: Control dextran injection


title: Dextran Control 24H sample 3


title: SPET-seq (Ribo- RNA), Replicate #2

description:
SPET-seq on rRNA-depleted RNA from nucleoid fraction
processed data file: SPET-seq.rc.tar.gz

data_processing:
library strategy: SPET-seq
FastQ files were examined using the FastQC tool. All the relevant SPET-seq data (nascent RNA) analysis and normalization steps were performed using a custom wrapper built on top of the RNA Framework13. Briefly, reads were clipped from 3’ adapter sequences using Cutadapt v1.10, discarding reads shorter than 15 nucleotides. Escherichia coli str. K-12 substr. MG1655 (GenBank: U00096.2) was used as the reference genome to extract transcripts’ sequences. Forward and reverse reads were independently mapped to the reference transcriptome using Bowtie v1.1.2, by allowing up to 7 mapping positions to enable mapping to the 7 E. coli rRNA genes (parameters: -n 2 -m 7 -a --best --strata -5 5 [--norc for forward reads, --nofw for reverse reads]). Forward and reverse mapped reads were then re-paired. Using reverse read mapping positions (corresponding to RNA Polymerase positions along gene), forward reads were split into separate SAM files for each transcription intermediate. When analysis was performed in deciles of transcription, genes were split into 10 equally sized deciles, and reads belonging to transcription intermediates falling in the same decile were pooled. SAM files were then passed to the rf-count tool of the RNA Framework to generate RT-stop counts (RC) files. Resulting RC files were normalized using the rf-norm tool of the RNA Framework in 50 nt sliding windows, with a 25 nt offset (parameters: -sm 2 -nm 2 -ec 0 -mc 0 -n 50 -nw 50 -wo 25). Mapping of DMS-seq data (mature RNA) was performed by using the rf-count tool (parameters: -cl 15 -bm 7 -ba -b5 5). Resulting RC files were normalized using the rf-norm tool (parameters: -sm 2 -nm 2 -ec 50 -mc 50 -n 50 -nw 50 -wo 25). The rf-norm tool generates a XML file for each transcript (or for each transcription intermediate/decile in the case of SPET-seq data). XML files for mature RNA were passed to the rf-fold tool of the RNA Framework (using ViennaRNA Package 2.2 with soft constraints14) to infer mature RNA structures (parameters: -md 600 -nlp).
Genome_build: U00096.2
Supplementary_files_format_and_content: Mapped reads were rescaled to a size of 10 bp, and BEDGraph files were generated using the genomeCoverageBed utility of the BEDTools suite.
Supplementary_files_format_and_content: DMS-seq data (pooled Total and Ribo- RNA) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data (pooled Total and Ribo- RNA from both replicates) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For each analyzed transcript, 10 files corresponding to SPET-seq data for the 10 transcription deciles are provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. A file corresponding to each transcription intermediate is provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format

organism: Escherichia coli

source_name: SPET-seq (Ribo- RNA)

extract_protocol:
Each bacteria pellet from a 25 ml culture (OD600 ~0.3) was homogeneously resuspended in 200 µl of Buffer A [10 mM Tris pH 8.0; 20% Sucrose; 100 mM NaCl] supplemented with 200 U SUPERase• In™ RNase Inhibitor, by pipetting. 50 µl of Buffer B [50 mM EDTA; 120 mM Tris pH 8.0] supplemented with 1 µl Ready-Lyse™ Lysozyme Solution (Epicentre, cat. R1810M) were added dropwise, and the vial was gently tilted 5 times to ensure homogenous mixing. The sample was then incubated 1 minute at room temperature. 250 µl of Buffer C [0.5% Tween-20: 0.4% NaDOC; 2 M NaCl; 10 mM EDTA] were immediately added dropwise. The sample was then incubated 5 minutes at room temperature. At this stage the solution clears considerably without increasing its viscosity, and nucleoid becomes visible. Using a cut P1000 pipette tip, the whole sample was gently layered on the top of a 5-30% w/v sucrose gradient [10 mM Tris pH 8.0; 1 M NaCl; 1 mM EDTA; 1 mM DTT], and centrifuged at 17,000 RPM in a SW55Ti rotor (Beckman Coulter, cat. 342194) for 9 minutes (4°C). After centrifugation, the nucleoid fraction was collected using a syringe with a 18G blunt fill needle, and transferred to a new centrifuge tube. The remaining gradient was assumed to represent the cytosolic fraction. The nucleoid was then resuspended in 2.5 ml Wash & Resuspension buffer [40 mM Tris pH 7.5; 150 mM KCl; 10 mM MgCl2; 1 mM DTT; 0.01% Triton X-100] supplemented with 200 U SUPERase• In™ RNase Inhibitor, pulse vortexed for 5 seconds, and then centrifuged at 28,000 RPM in a SW55Ti rotor for 30 minutes (0°C). After centrifugation the supernatant was decanted, and the nucleoid pellet was washed twice with 2 ml of Wash & Resuspension buffer, taking care not to disturb it. The nucleoid was then resuspended in 500 µl Wash & Resuspension buffer, and solubilized by addition of 0.1 gr acid-washed glass beads (Sigma, cat. G1145), and shaking for 5 minutes in a TissueLyser (QIAGEN). For each 100 µl of purified nucleoids (or cytosolic fraction), 1 ml of TRIzol® Reagent (Invitrogen, cat. 15596-018) was added, and RNA was extracted following manufacturer’s instructions. RNA was analyzed on a 2100 Bioanalyzer (Agilent). In all experiments, RNA from cytosolic fraction (corresponding to mature RNA species) had RIN > 9.5. Total RNA yield from nucleoid fraction was ~6% of the total RNA content.
10 pmol of a pre-adenylated (rApp) adapter were ligated to 1 μg of nascent RNA (either total, or rRNA-depleted using Ribo-Zero rRNA Removal Kit (Illumina, cat. MRZB12424)) in a reaction volume of 20 μl, using 400 U T4 RNA Ligase 2, Deletion Mutant in the presence of 20% PEG-8000, by incubation at 25°C for 2 hours. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and RNA was eluted in 5.5 μl nuclease-free water. RNA was heat-denatured at 70°C for 5 minutes, and reverse transcription was carried out in a final volume of 10 μl, in the presence of 0.5 mM dNTPs, 5 pmol of RT primer, 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, and 100 U SuperScript® III Reverse Transcriptase, by incubation at 50°C for 50 minutes. Template RNA was degraded by adding 1 μl of 1 M NaOH, and incubating at 95°C for 5 minutes. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and cDNA was eluted in 6 μl nuclease-free water. cDNA fragments were resolved on a 10% TBE-Urea polyacrylamide gel, and three gel slices corresponding to fragments in the ranges of 40-200 nt, 200-400 nt, and 400-600 nt were cut. DNA was recovered by passive diffusion in Diffusion buffer for 16 hours at 37°C with moderate shaking. cDNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 8.25 μl nuclease-free water. 10 pmol of a 5’-phosphorilated adapter were ligated to the 3’-OH of cDNA fragments in a final reaction volume of 25 μl, in the presence of 0.05 mM ATP, 20% PEG-4000, and 100 U CircLigase™ II ssDNA Ligase, by incubation at 60°C for 4 hours, and 68°C for 2 hours. Adapter-ligated cDNA fragments were purified from excess adapter using 1.8 volumes of Agencourt AMPure XP beads, following manufacturer’s instructions. cDNA was eluted in 20 μl of nuclease-free water, and indexed sequencing adapters were introduced by 15 cycles of PCR in the presence of 25 pmol of each primer, and 25 μl NEBNext® High-Fidelity 2X PCR Master Mix.

treatment_protocol: A single colony of Escherichia coli was inoculated into 250 ml of LB medium without antibiotics, and grown at 37C with shaking (150 RPM) for approximately 4 hours, until OD600 was ~0.3 (log phase). EDTA was then added to a final concentration of 1 mM, and incubated for additional 5 minutes to make bacteria permeable to Actinomycin D. Actinomycin D was then added to a final concentration of 5  g/ml, and bacteria were incubated for additional 2 minutes to block transcription. 25 ml aliquots of bacteria were then rapidly pelleted by centrifugation at 4C. After centrifugation, medium was decanted, and cells from each 25ml of culture were resuspended in 1ml of structure probing buffer [10 mM HEPES-KOH pH 7.9; 140 mM NaCl; 3 mM KCl]. DMS was diluted 1:6 in 100% Ethanol to a final concentration of 1.76 M. Diluted DMS was added to bacteria to a final concentration of ~105 mM. Samples were incubated with moderate shaking (800 RPM) at 25C for 2 minutes, after which reactions were immediately transferred to ice. DTT was added to a final concentration of 0.7 M to quench DMS, and samples were vigorously vortexed for 10 seconds. Bacteria were then pelleted by centrifugation at 10,000g for 30 seconds (4C), and the supernatant was decanted. Pellets were then washed once with 1 ml Isoamyl alcohol (Sigma Aldrich, cat. W205702) to remove traces of water-insoluble DMS. Bacteria were then pelleted by additional centrifugation at 10,000g for 30 seconds (4C), supernatant was decanted, and samples were snap-frozen in liquid nitrogen. Pellets were stored at -80C.

tag: substr

tag: molecule subtype

value: Total RNA (Nucleoid fraction)


title: col0_leaf2.sRNAseq

description: Single-end 50 base read small RNA-Seq data from second biological replicate of Col-0 leaves


title: col0_fb1.sRNAseq

description: Single-end 50 base read small RNA-Seq data from first biological replicate of Col-0 flowers


title: Dextran Control 24H sample 1

tag: treatment


title: OSR2 RNA Rep2

value: Lohmann Selected Leghorn

value: limb buds


title: col0_leaf2.mRNAseq

description:
Paired-end 50 base read poly(A) RNA-Seq data from second biological replicate of Col-0 leaves
Processed data is Series supplementary file master_genes_Araport.txt


title: SPET-seq (Ribo- RNA), Replicate #1


title: DMS-seq (Ribo- RNA)

description:
DMS-seq on cytosolic RNA fraction
processed data file: DMS-seq.rc.tar.gz

data_processing:
library strategy: DMS-seq
FastQ files were examined using the FastQC tool. All the relevant SPET-seq data (nascent RNA) analysis and normalization steps were performed using a custom wrapper built on top of the RNA Framework13. Briefly, reads were clipped from 3’ adapter sequences using Cutadapt v1.10, discarding reads shorter than 15 nucleotides. Escherichia coli str. K-12 substr. MG1655 (GenBank: U00096.2) was used as the reference genome to extract transcripts’ sequences. Forward and reverse reads were independently mapped to the reference transcriptome using Bowtie v1.1.2, by allowing up to 7 mapping positions to enable mapping to the 7 E. coli rRNA genes (parameters: -n 2 -m 7 -a --best --strata -5 5 [--norc for forward reads, --nofw for reverse reads]). Forward and reverse mapped reads were then re-paired. Using reverse read mapping positions (corresponding to RNA Polymerase positions along gene), forward reads were split into separate SAM files for each transcription intermediate. When analysis was performed in deciles of transcription, genes were split into 10 equally sized deciles, and reads belonging to transcription intermediates falling in the same decile were pooled. SAM files were then passed to the rf-count tool of the RNA Framework to generate RT-stop counts (RC) files. Resulting RC files were normalized using the rf-norm tool of the RNA Framework in 50 nt sliding windows, with a 25 nt offset (parameters: -sm 2 -nm 2 -ec 0 -mc 0 -n 50 -nw 50 -wo 25). Mapping of DMS-seq data (mature RNA) was performed by using the rf-count tool (parameters: -cl 15 -bm 7 -ba -b5 5). Resulting RC files were normalized using the rf-norm tool (parameters: -sm 2 -nm 2 -ec 50 -mc 50 -n 50 -nw 50 -wo 25). The rf-norm tool generates a XML file for each transcript (or for each transcription intermediate/decile in the case of SPET-seq data). XML files for mature RNA were passed to the rf-fold tool of the RNA Framework (using ViennaRNA Package 2.2 with soft constraints14) to infer mature RNA structures (parameters: -md 600 -nlp).
Genome_build: U00096.2
Supplementary_files_format_and_content: Mapped reads were rescaled to a size of 10 bp, and BEDGraph files were generated using the genomeCoverageBed utility of the BEDTools suite.
Supplementary_files_format_and_content: DMS-seq data (pooled Total and Ribo- RNA) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data (pooled Total and Ribo- RNA from both replicates) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For each analyzed transcript, 10 files corresponding to SPET-seq data for the 10 transcription deciles are provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. A file corresponding to each transcription intermediate is provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format

source_name: DMS-seq (Ribo- RNA)

extract_protocol:
Each bacteria pellet from a 25 ml culture (OD600 ~0.3) was homogeneously resuspended in 200 µl of Buffer A [10 mM Tris pH 8.0; 20% Sucrose; 100 mM NaCl] supplemented with 200 U SUPERase• In™ RNase Inhibitor, by pipetting. 50 µl of Buffer B [50 mM EDTA; 120 mM Tris pH 8.0] supplemented with 1 µl Ready-Lyse™ Lysozyme Solution (Epicentre, cat. R1810M) were added dropwise, and the vial was gently tilted 5 times to ensure homogenous mixing. The sample was then incubated 1 minute at room temperature. 250 µl of Buffer C [0.5% Tween-20: 0.4% NaDOC; 2 M NaCl; 10 mM EDTA] were immediately added dropwise. The sample was then incubated 5 minutes at room temperature. At this stage the solution clears considerably without increasing its viscosity, and nucleoid becomes visible. Using a cut P1000 pipette tip, the whole sample was gently layered on the top of a 5-30% w/v sucrose gradient [10 mM Tris pH 8.0; 1 M NaCl; 1 mM EDTA; 1 mM DTT], and centrifuged at 17,000 RPM in a SW55Ti rotor (Beckman Coulter, cat. 342194) for 9 minutes (4°C). After centrifugation, the nucleoid fraction was collected using a syringe with a 18G blunt fill needle, and transferred to a new centrifuge tube. The remaining gradient was assumed to represent the cytosolic fraction. The nucleoid was then resuspended in 2.5 ml Wash & Resuspension buffer [40 mM Tris pH 7.5; 150 mM KCl; 10 mM MgCl2; 1 mM DTT; 0.01% Triton X-100] supplemented with 200 U SUPERase• In™ RNase Inhibitor, pulse vortexed for 5 seconds, and then centrifuged at 28,000 RPM in a SW55Ti rotor for 30 minutes (0°C). After centrifugation the supernatant was decanted, and the nucleoid pellet was washed twice with 2 ml of Wash & Resuspension buffer, taking care not to disturb it. The nucleoid was then resuspended in 500 µl Wash & Resuspension buffer, and solubilized by addition of 0.1 gr acid-washed glass beads (Sigma, cat. G1145), and shaking for 5 minutes in a TissueLyser (QIAGEN). For each 100 µl of purified nucleoids (or cytosolic fraction), 1 ml of TRIzol® Reagent (Invitrogen, cat. 15596-018) was added, and RNA was extracted following manufacturer’s instructions. RNA was analyzed on a 2100 Bioanalyzer (Agilent). In all experiments, RNA from cytosolic fraction (corresponding to mature RNA species) had RIN > 9.5. Total RNA yield from nucleoid fraction was ~6% of the total RNA content.
2 μg of RNA from the cytoplasmic fraction were diluted in 20 μl of Fragmentation buffer, and fragmented by incubation at 95°C for 5 minutes. Fragmented RNA was purified using RNA Clean & Concentrator™-5 columns. End repair of RNA fragments was performed in a final volume of 20 μl, in the presence of 20 U T4 Polynucleotide Kinase (NEB, cat. M0201L), and 20 U SUPERase• In™ RNase Inhibitor, by incubation at 37°C for 1 hour. End-repaired RNA was purified again using RNA Clean & Concentrator™-5 columns, and eluted in 6 μl of nuclease-free water. 6 μl of 2X RNA Loading Dye were added to end-repaired RNA. RNA was heated to 95°C for 2 minutes, and immediately placed on ice. Samples were resolved on a 10% TBE-Urea polyacrylamide gel, and a gel slice corresponding to fragments above 200 nt was cut. The gel slice was crushed by centrifugation through a punctured 0.5 ml tube, and resuspended in 500 μl of Diffusion buffer supplemented with 60 U SUPERase• In™ RNase Inhibitor, then rotated at 4°C for 16 hours to allow passive diffusion of RNA fragments into buffer. RNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 6 μl nuclease-free water. 10 pmol of a pre-adenylated (rApp) adapter were ligated to size-selected RNA fragments in a reaction volume of 20 μl, using 400 U T4 RNA Ligase 2, Deletion Mutant in the presence of 20% PEG-8000, by incubation at 25°C for 2 hours. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and RNA was eluted in 5.5 μl nuclease-free water. RNA was heat-denatured at 70°C for 5 minutes, and reverse transcription was carried out in a final volume of 10 μl, in the presence of 0.5 mM dNTPs, 5 pmol of RT primer, 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, and 100 U SuperScript® III Reverse Transcriptase, by incubation at 50°C for 50 minutes. Template RNA was degraded by adding 1 μl of 1 M NaOH, and incubating at 95°C for 5 minutes. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and cDNA was eluted in 6 μl nuclease-free water. cDNA fragments were resolved on a 10% TBE-Urea polyacrylamide gel, and a gel slice corresponding to fragments in the range of 40-150 nt was cut (corresponding to truncated cDNA products). DNA was recovered by passive diffusion in Diffusion buffer for 16 hours at 37°C with moderate shaking. cDNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 8.25 μl nuclease-free water. 10 pmol of a 5’-phosphorilated adapter were ligated to the 3’-OH of cDNA fragments in a final reaction volume of 25 μl, in the presence of 0.05 mM ATP, 20% PEG-4000, and 100 U CircLigase™ II ssDNA Ligase, by incubation at 60°C for 4 hours, and 68°C for 2 hours. Adapter-ligated cDNA fragments were purified from excess adapter using 1.8 volumes of Agencourt AMPure XP beads, following manufacturer’s instructions. cDNA was eluted in 20 μl of nuclease-free water, and indexed sequencing adapters were introduced by 15 cycles of PCR in the presence of 25 pmol of each primer, and 25 μl NEBNext® High-Fidelity 2X PCR Master Mix.

value: Total RNA (Cytosolic fraction)


title: SPET-seq (Total RNA)

description:
SPET-seq on total RNA from nucleoid fraction
processed data file: SPET-seq.rc.tar.gz

data_processing:
library strategy: SPET-seq
FastQ files were examined using the FastQC tool. All the relevant SPET-seq data (nascent RNA) analysis and normalization steps were performed using a custom wrapper built on top of the RNA Framework13. Briefly, reads were clipped from 3’ adapter sequences using Cutadapt v1.10, discarding reads shorter than 15 nucleotides. Escherichia coli str. K-12 substr. MG1655 (GenBank: U00096.2) was used as the reference genome to extract transcripts’ sequences. Forward and reverse reads were independently mapped to the reference transcriptome using Bowtie v1.1.2, by allowing up to 7 mapping positions to enable mapping to the 7 E. coli rRNA genes (parameters: -n 2 -m 7 -a --best --strata -5 5 [--norc for forward reads, --nofw for reverse reads]). Forward and reverse mapped reads were then re-paired. Using reverse read mapping positions (corresponding to RNA Polymerase positions along gene), forward reads were split into separate SAM files for each transcription intermediate. When analysis was performed in deciles of transcription, genes were split into 10 equally sized deciles, and reads belonging to transcription intermediates falling in the same decile were pooled. SAM files were then passed to the rf-count tool of the RNA Framework to generate RT-stop counts (RC) files. Resulting RC files were normalized using the rf-norm tool of the RNA Framework in 50 nt sliding windows, with a 25 nt offset (parameters: -sm 2 -nm 2 -ec 0 -mc 0 -n 50 -nw 50 -wo 25). Mapping of DMS-seq data (mature RNA) was performed by using the rf-count tool (parameters: -cl 15 -bm 7 -ba -b5 5). Resulting RC files were normalized using the rf-norm tool (parameters: -sm 2 -nm 2 -ec 50 -mc 50 -n 50 -nw 50 -wo 25). The rf-norm tool generates a XML file for each transcript (or for each transcription intermediate/decile in the case of SPET-seq data). XML files for mature RNA were passed to the rf-fold tool of the RNA Framework (using ViennaRNA Package 2.2 with soft constraints14) to infer mature RNA structures (parameters: -md 600 -nlp).
Genome_build: U00096.2
Supplementary_files_format_and_content: Mapped reads were rescaled to a size of 10 bp, and BEDGraph files were generated using the genomeCoverageBed utility of the BEDTools suite.
Supplementary_files_format_and_content: DMS-seq data (pooled Total and Ribo- RNA) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data (pooled Total and Ribo- RNA from both replicates) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For each analyzed transcript, 10 files corresponding to SPET-seq data for the 10 transcription deciles are provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. A file corresponding to each transcription intermediate is provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format. For rRNA 23S (rrlB gene), SPET-seq data is provided in the form of RC files, each one corresponding to individual transcription intermediates.

source_name: SPET-seq (Total RNA)


title: DnTCF 24H sample 2

value: RNA injection of the dominant negative form of NvTCF


title: atrx-1_2

data_processing:
Image analysis and base calling were performed using RTA 1.18.61 and CASAVA 1.8.2.
DeSeq2
After removal of rRNA reads, reads were mapped onto the TAIR10 genome assembly of Arabidopsis thaliana using TopHat v2.0.14 and Bowtie v2.1.0. Gene expression quantification was performed from uniquely aligned reads using HTSeq-count v0.6.1 (intersection-non empty mode). DESeq2 v1.6.3 was used to normalize read counts and evaluate statistical significance of differential expression with cooksCutoff option set to false.
Genome_build: TAIR10
Supplementary_files_format_and_content: The excel file display results for HTSeq read counts for each gene and the statistical analysis results obtained with DeSeq.

source_name: atrx-1 mutant_14d-old in vitro grown plantlets

growth_protocol: 14d-old in vitro grown plantlets

extract_protocol:
Trizol followed by Plant Ribominus kit
Library prep for total RNA Ribozero

tag: ecotype background

value: col

value: atrx-1 mutant

tag: age

value: seedlings; in vitro grown plantlets


title: DnTCF 24H sample 1


title: DMS-seq (Total RNA)

source_name: DMS-seq (Total RNA)


title: atrx-1_1


title: 3'-end RNA-seq, Nucleoid fraction, Replicate #2

description:
3'-end RNA-seq on nucleoid RNA fraction
3'-end RNA-seq Nucleoid #2

data_processing:
library strategy: 3'-end RNA-seq
FastQ files were examined using the FastQC tool. Reads were clipped from 3’ adapter sequences using Cutadapt v1.10, discarding reads shorter than 15 nucleotides. Escherichia coli str. K-12 substr. MG1655 (GenBank: U00096.2) was used as the reference genome. Reads were mapped to the reference genome using Bowtie v1.1.2, by allowing up to 7 mapping positions to enable mapping to the 7 E. coli rRNA genes (parameters: -n 2 -m 7 -a --best --strata -5 5).
Genome_build: U00096.2
Supplementary_files_format_and_content: Mapped reads were rescaled to a size of 10 bp, and BEDGraph files were generated using the genomeCoverageBed utility of the BEDTools suite.
Supplementary_files_format_and_content: DMS-seq data (pooled Total and Ribo- RNA) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data (pooled Total and Ribo- RNA from both replicates) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For each analyzed transcript, 10 files corresponding to SPET-seq data for the 10 transcription deciles are provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. A file corresponding to each transcription intermediate is provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format

source_name: 3'-end RNA-seq, Nucleoid fraction

extract_protocol:
Each bacteria pellet from a 25 ml culture (OD600 ~0.3) was homogeneously resuspended in 200 µl of Buffer A [10 mM Tris pH 8.0; 20% Sucrose; 100 mM NaCl] supplemented with 200 U SUPERase• In™ RNase Inhibitor, by pipetting. 50 µl of Buffer B [50 mM EDTA; 120 mM Tris pH 8.0] supplemented with 1 µl Ready-Lyse™ Lysozyme Solution (Epicentre, cat. R1810M) were added dropwise, and the vial was gently tilted 5 times to ensure homogenous mixing. The sample was then incubated 1 minute at room temperature. 250 µl of Buffer C [0.5% Tween-20: 0.4% NaDOC; 2 M NaCl; 10 mM EDTA] were immediately added dropwise. The sample was then incubated 5 minutes at room temperature. At this stage the solution clears considerably without increasing its viscosity, and nucleoid becomes visible. Using a cut P1000 pipette tip, the whole sample was gently layered on the top of a 5-30% w/v sucrose gradient [10 mM Tris pH 8.0; 1 M NaCl; 1 mM EDTA; 1 mM DTT], and centrifuged at 17,000 RPM in a SW55Ti rotor (Beckman Coulter, cat. 342194) for 9 minutes (4°C). After centrifugation, the nucleoid fraction was collected using a syringe with a 18G blunt fill needle, and transferred to a new centrifuge tube. The remaining gradient was assumed to represent the cytosolic fraction. The nucleoid was then resuspended in 2.5 ml Wash & Resuspension buffer [40 mM Tris pH 7.5; 150 mM KCl; 10 mM MgCl2; 1 mM DTT; 0.01% Triton X-100] supplemented with 200 U SUPERase• In™ RNase Inhibitor, pulse vortexed for 5 seconds, and then centrifuged at 28,000 RPM in a SW55Ti rotor for 30 minutes (0°C). After centrifugation the supernatant was decanted, and the nucleoid pellet was washed twice with 2 ml of Wash & Resuspension buffer, taking care not to disturb it. The nucleoid was then resuspended in 500 µl Wash & Resuspension buffer, and solubilized by addition of 0.1 gr acid-washed glass beads (Sigma, cat. G1145), and shaking for 5 minutes in a TissueLyser (QIAGEN). For each 100 µl of purified nucleoids (or cytosolic fraction), 1 ml of TRIzol® Reagent (Invitrogen, cat. 15596-018) was added, and RNA was extracted following manufacturer’s instructions. RNA was analyzed on a 2100 Bioanalyzer (Agilent). In all experiments, RNA from cytosolic fraction (corresponding to mature RNA species) had RIN > 9.5. Total RNA yield from nucleoid fraction was ~6% of the total RNA content.
10 pmol of a pre-adenylated (rApp) adapter were ligated to 1 μg of nascent (or mature) RNA in a reaction volume of 20 μl, using 400 U T4 RNA Ligase 2, Deletion Mutant (Epicentre, cat. LR2D11310K) in the presence of 20% PEG-8000, by incubation at 25°C for 2 hours. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and RNA was eluted in 20 μl of Fragmentation buffer [65 mM Tris pH 8.3; 100 mM KCl; 5 mM MgCl2]. RNA was fragmented by incubation at 95°C for 8 minutes. Fragmented RNA was purified using RNA Clean & Concentrator™-5 columns, and eluted in 5.5 μl of nuclease-free water. RNA was heat-denatured at 70°C for 5 minutes, and reverse transcription was carried out in a final volume of 10 μl, in the presence of 0.5 mM dNTPs, 5 pmol of RT primer, 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen, cat. 10777-019), and 100 U SuperScript® III Reverse Transcriptase (Invitrogen, cat. 18080-044), by incubation at 50°C for 50 minutes. Template RNA was degraded by adding 1 μl of 1 M NaOH, and incubating at 95°C for 5 minutes. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and cDNA was eluted in 6 μl nuclease-free water. cDNA fragments were resolved on a 10% TBE-Urea polyacrylamide gel, and a gel slice corresponding to fragments in the range 40-150 nt was cut. DNA was recovered by passive diffusion in Diffusion buffer for 16 hours at 37°C with moderate shaking. cDNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 8.25 μl nuclease-free water. 10 pmol of a 5’-phosphorylated adapter were ligated to the 3’-OH of cDNA fragments in a final reaction volume of 25 μl, in the presence of 0.05 mM ATP, 20% PEG-4000, and 100 U CircLigase™ II ssDNA Ligase (Epicentre, cat. CL9025K), by incubation at 60°C for 4 hours, and 68°C for 2 hours. Adapter-ligated cDNA fragments were purified from excess adapter using 1.8 volumes of Agencourt AMPure XP beads (Beckman Coulter, cat. A63881), following manufacturer’s instructions. cDNA was eluted in 20 μl of nuclease-free water, and indexed sequencing adapters were introduced by 15 cycles of PCR in the presence of 25 pmol of each primer, and 25 μl NEBNext® High-Fidelity 2X PCR Master Mix (NEB, cat. M0541L).


title: BMP24 24H KD sample 3

value: Morpholino knockdown of NvBMP2/4


title: col_3

source_name: WT_14d-old in vitro grown plantlets

value: wild type


title: Control RNA Rep2

value: mesenchymal cells

value: 5 days

value: RCAS-BP(A)-empty


title: col0_fb2.sRNAseq

description: Single-end 50 base read small RNA-Seq data from second biological replicate of Col-0 flowers

value: unopened floral buds


title: BMP24 24H KD sample 2


title: 3'-end RNA-seq, Nucleoid fraction, Replicate #1

description:
3'-end RNA-seq on nucleoid RNA fraction
3'-end RNA-seq Nucleoid #1


title: col_2


title: BMP24 24H KD sample 1


title: col_1


title: 3'-end RNA-seq, Cytosolic fraction, Replicate #2

description:
3'-end RNA-seq on cytosolic RNA fraction
3'-end RNA-seq Cytosol #2

source_name: 3'-end RNA-seq, Cytosolic fraction


title: atrx-1_3


title: DnTCF 24H sample 3


title: col0_leaf1.sRNAseq

description: Single-end 50 base read small RNA-Seq data from first biological replicate of Col-0 leaves

value: rosette and cauline leaves


title: 3'-end RNA-seq, Cytosolic fraction, Replicate #1

description:
3'-end RNA-seq on cytosolic RNA fraction
3'-end RNA-seq Cytosol #1


title: met1 sRNA

status: Public on Jul 18 2017

city: San Francisco

first: Stefan

last: Oberlin

address: 505 Parnassus Ave

department: Department of Microbiology and Immunology

institute: University of California, San Francisco

data_processing:
Illumina Casava1.8 software used for basecalling.
Trimming of reads with Trimmomatic-0-2.36 using the following parameters: SLIDINGWINDOW:4:10 AVGQUAL:20 MINLEN:50
Mapping to Arabidopsis genome (TAIR10) with STAR (--outFilterMultimapNmax 50 --outFilterMatchNmin 30 --alignSJoverhangMin 3 --alignIntronMax 10000)
Read counting with Rsubread (allowMultiOverlap=T, isPairedEnd=T, strandSpecific=2, countMultiMappingReads=T)
Read normalization with edgeR.
sRNA libraries were trimmed with Trimmomatic-0-2.36 using the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:15
sRNA counting was performed using the processReads-median.pl function of the ncPRO-seq v1.5.1 pipeline.
Genome_build: TAIR10
Supplementary_files_format_and_content: *_CPM.txt: Tab-delimited text files of count per million (CPM) values.
Supplementary_files_format_and_content: met1_sRNA_processed.txt: Tab-delimited text file of counts per sRNA species.

source_name: Inflorescence tissue

growth_protocol: Arabidopsis thaliana plants were grown on soil in a growth chamber at 22°C for two weeks in a 12h/12h light cycle and then transferred to a 16h/8h light cycle.

extract_protocol:
RNA was extracted from inflorescence tissue using the TRIzol extraction method.
To generate total RNA-seq libraries, RNA was subjected to ribodepletion with Ribo Zero (Illumina) and libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina). RNA 3’end libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen) adjusted for longer insert lengths by diluting the second strand synthesis buffer with water in a ratio 1:1. sRNA-seq libraries were constructed by Fasteris SA. Libraries were prepared using the Illumina TruSeq Kit SBS v3.

tag: rna type

value: small RNA

tag: library type

value: TruSeq sRNA


title: QuantSeq 3'end Polysome ddm1 Rep2

description:
Polysome_ddm1_2
QuantSeq_CPM.txt

tag: ecotype

tag: genotype/variation

value: polysome associated RNA

value: QuantSeq 3'end mRNA-seq


title: QuantSeq 3'end Polysome Col-0 Rep2

description:
Polysome_Col_2
QuantSeq_CPM.txt

value: wildtype


title: QuantSeq 3'end met1 Rep1

description:
met1_1
QuantSeq_CPM.txt

value: total RNA


title: QuantSeq 3'end Polysome Col-0 Rep1

description:
Polysome_Col_1
QuantSeq_CPM.txt


title: QuantSeq 3'end Polysome ddm1 Rep1

description:
Polysome_ddm1_1
QuantSeq_CPM.txt


title: QuantSeq 3'end met1 Rep2

description:
met1_2
QuantSeq_CPM.txt


title: QuantSeq 3'end ddm1 Rep2

description:
ddm1_2
QuantSeq_CPM.txt


title: TruSeq ddm1 Rep2

description:
ddm1_total2
TrueSeq_CPM.txt

value: TruSeq


title: QuantSeq 3'end Col-0 Rep1

description:
Col_1
QuantSeq_CPM.txt


title: TruSeq met1 Rep2

description:
met1_total2
TrueSeq_CPM.txt


title: TruSeq met1 Rep1

description:
met1_total1
TrueSeq_CPM.txt


title: TruSeq ddm1 Rep1

description:
ddm1_total1
TrueSeq_CPM.txt


title: TruSeq Col-0 Rep2

description:
Col0_total2
TrueSeq_CPM.txt


title: TruSeq Col-0 Rep1

description:
Col0_total1
TrueSeq_CPM.txt


title: WT.r3_ATAC_bio1

first: Pascale

last: Gilardi-Hebenstreit

address: 46 Rue d'Ulm

department: IBENS - UMR ENS - CNRS 8197 - INSERM 1024

institute: Institut de Biologie de l'Ecole Normale Superieure

description: CharnayLab_WT.r3_ATAC_bio1_160601_run1_Aligned.sortedByCoord.out.rmdup.frag100max.bw

data_processing:
paired-end reads were mapped onto the mouse genome assembly mm9 using STAR (outFilterMultimapNmax 1; outFilterMismatchNmax 999; outFilterMismatchNoverLmax 0.06 ; alignIntronMax 1; alignEndsType EndToEnd; alignMatesGapMax 2000).
Duplicates reads were removed using Picard (http://picard.sourceforge.net) (MarkDuplicates, REMOVE_DUPLICATES = true).
Bigwig tracks were made using DeepTools BamCoverage (1.5.9.1), keeping only fragment smaller than 100bp to exclude fragment containing nucleosomes.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig of signal from fragments < 100bp

source_name: E8.5 embryo_3rd rhombomere

growth_protocol: All mouse lines were maintained in a mixed C57BL6/DBA2 background. We used the following alleles: Krox20Cre (Voiculescu et al. 2000) and Krox20∆A (Bouchoucha et al. 2013). The mouse Krox20CFlox line was generated at the Institut Clinique de la Souris (Illkirch, France) by homologous recombination in ES cells. The Krox20∆C allele was obtained as described in Figure 1. All animal manipulations were approved by a French Ethical Committee (Project Ce5/ 2012/120) and were performed according to French and European Union regulations.

extract_protocol:
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. ATAC experiments were performed according to Buenrostros et al., (2013) protocol and using a homemade transposome from Picelli et al., (2014). 7-8 embryos at 10-12s were dissected in cold PBS for each genotype and cells were mechanically dissociated. Cells were lysed before transposition using 1µL of transposome and purified using a Qiagen MinElute Kit with 10µL of Elution Buffer. Transposed DNA was amplified by PCR as previously described and quantified by qPCR using 5µL of PCR products. The number of additionnal cycles is determined by plotting linear Rn versus cycle and correspond to the one-third of the maximum fluorescence intensity. The remaining 45µL of PCR products are runned with the additional number of cycles. The final product is purified with Qiagen PCR Cleanup Kit and eluted in 20µL Elution Buffer.
Sequencing was done on multiplexed samples using 42 bp Paired-end reads on the Illumina NextSeq system according to the manufacturer’s specifications at the genomics platform of IBENS.

tag: strain background

value: WT

tag: tissue

value: 3rd rhombomere


title: WT.r5_ATAC_bio1

description: CharnayLab_WT.r5_ATAC_bio1_160601_run1_Aligned.sortedByCoord.out.rmdup.frag100max.bw

source_name: E8.5 embryo_5th rhombomere

value: 5th rhombomere


title: WT.r5_ATAC_bio2

description: CharnayLab_WT.r5_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: WT.post_ATAC_bio2

description: CharnayLab_WT.post_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw

source_name: E8.5 embryo_posterior part

value: posterior part


title: WT.r3_ATAC_bio2

description: CharnayLab_WT.r3_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: LT_old_mouse2_plate1_stim_sc_C1_S313

data_processing:
Mapping reads to transcriptome with RSEM
Flag cells due to: not a single cell (100 cells; pop); low percent of reads mapping to transcriptome (pctTrans); low number of genes detected (fewGenes); cells undergoing cell cycle (inferred from expression signature; cycling); contaminating plasma cell, based on gene expression signature (plasma)
Genome_build: mm9
Supplementary_files_format_and_content: TPM and raw counts for all single cell samples

source_name: HSCs identified by cell surface markers, isolated from mouse bone marrow.

extract_protocol:
Single cell and 100 cell populations were lysed by sorting into 5ul TCL Buffer (QIAGEN) with 1% 2-Mercaptoethanol, centrifuged and frozen at -80°C - this was used for SMART-seq2 as described in Picelli et al. Nat. Prot. 2014.
SMART-seq2

value: LT

tag: mouse age

value: old

tag: mouse id

value: LPS+PAM stimulation

tag: plate id

tag: well id

tag: sample type

value: single cell


title: WT.post_ATAC_bio1

description: CharnayLab_WT.post_ATAC_bio1_160601_run1_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: WT.E9.5.r5_ATAC_bio2

description: CharnayLab_WT.E9.5.r5_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw

source_name: E9.5 embryo_5th rhombomere


title: WT.E9.5.post_ATAC_bio2

description: CharnayLab_WT.E9.5.post_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw

source_name: E9.5 embryo_posterior part


title: WT.E9.5.r3_ATAC_bio2

description: CharnayLab_WT.E9.5.r3_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw

source_name: E9.5 embryo_3rd rhombomere


title: WT.E9.5.r3_ATAC_bio1

description: CharnayLab_WT.E9.5.r3_ATAC_bio1_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: QuantSeq 3'end ddm1 Rep1

description:
ddm1_1
QuantSeq_CPM.txt


title: QuantSeq 3'end Col-0 Rep2

description:
Col_2
QuantSeq_CPM.txt


title: deltaC.r5_ATAC_bio1

description: CharnayLab_deltaC.r5_ATAC_bio1_160601_run1_Aligned.sortedByCoord.out.rmdup.frag100max.bw

value: deltaC


title: DeltaA.r5_ATAC_bio1

description: CharnayLab_DeltaA.r5_ATAC_bio1_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw

value: deltaA


title: DeltaC.r3_ATAC_bio2

description: CharnayLab_DeltaC.r3_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: deltaC.r3_ATAC_bio1

description: CharnayLab_deltaC.r3_ATAC_bio1_160601_run1_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: deltaC.post_ATAC_bio1

description: CharnayLab_deltaC.post_ATAC_bio1_160601_run1_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: DeltaC.post_ATAC_bio2

description: CharnayLab_DeltaC.post_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: DeltaA.r5_ATAC_bio2

description: CharnayLab_DeltaA.r5_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: DeltaA.post_ATAC_bio2

description: CharnayLab_DeltaA.post_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: DeltaA.r3_ATAC_bio1

description: CharnayLab_DeltaA.r3_ATAC_bio1_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: DeltaA.r3_ATAC_bio2

description: CharnayLab_DeltaA.r3_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: WT.E9.5.r5_ATAC_bio1

description: CharnayLab_WT.E9.5.r5_ATAC_bio1_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: 24h LG268 amd GW3965 RNA-seq in mES Rep2

city: Orlando

first: Laszlo

last: Nagy

state: Hungary

address: Sanger Road

institute: Sandford Burnham Prebys Institute

data_processing:
alignment was performed  with TopHat v2.0.7 using default parameters.
RPKM values were calculated by cufflinks v2.0.2 using default parameters.
Raw counts were calculated by by HTSeq 0.6.0 using default parameters.
Statististical analysis was performed using cuffdiff v2.0.2 (RPKM>1 at least in one condition, p-value<=0.05, FC>2).
Genome_build: mm10

source_name: embryonic stem cells

growth_protocol: Wild type E14 mouse ESCs were cultured on 0.1% gelatin coated plates in feeder-free condition in 5% CO2 at 37˚C. The ESC medium was prepared by supplementing DMEM Glutamax media with 15% FBS (Hyclone), 1000U of leukemia inhibitory factor (LIF), penicillin/streptomycin, non-essential amino acids and 2-mercaptoethanol. Cells were differentiated through embryoid body formation (Bibel et al., 2007). Briefly, for the induction of EB formation cells were aggregated in 100-mm bacteriological dishes (Greiner) at a specific density (4x106 cells/ 15 ml) in the absence of LIF. Over the span of four days, neural differentiation was induced by all-trans retinoic acid (RA) (Sigma) in 5 µM final concentration. After  four days of propagation trypsinized EBs were plated onto laminin (Sigma)/ poly-L-ornithine (Sigma) precoated plates at a density of 1 x 105 cells / cm2 in N2 medium (Bibel et al., 2007).

extract_protocol:
RNA was harvested using Trizol reagent
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cells were treated with vehicle (dimethylsulfoxide) or with the following ligands in 1µM final concentration: RA (Sigma), LG268 (Boehm et al., 1995) (a gift from R. Heyman; Ligand Pharmaceuticals, San Diego, CA), GW3965 (Sigma). For dose response analyses, ligands were used at the indicated concentrations.

tag: cell type

value: embryonic stem cells


title: 24h LG268 RNA-seq in mES Rep2


title: 24h LG268 amd GW3965 RNA-seq in mES Rep1


title: 24h LG268 RNA-seq in mES Rep1


title: 24h GW3965 RNA-seq in mES Rep2


title: WT.E9.5.post_ATAC_bio1

description: CharnayLab_WT.E9.5.post_ATAC_bio1_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: DeltaC.r5_ATAC_bio2

description: CharnayLab_DeltaC.r5_ATAC_bio2_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: 24h GW3965 RNA-seq in mES Rep1


title: Day 17 GW3965 RNA-seq in mES Rep1


title: Day 17 RNA-seq in mES Rep1

value: day 17


title: Day 8 RNA-seq in mES Rep2

value: day 8


title: Day 8 RNA-seq in mES Rep1


title: Day 16 GW3965 RNA-seq in mES Rep1


title: Day 16 RNA-seq in mES Rep2

value: day 16


title: Day 16 RNA-seq in mES Rep1


title: DeltaA.post_ATAC_bio1

description: CharnayLab_DeltaA.post_ATAC_bio1_160729_Aligned.sortedByCoord.out.rmdup.frag100max.bw


title: CTRL RNA-seq in mES Rep1

description: control for LG and GW treatment

value: day 0


title: CTRL RNA-seq in mES Rep2

description: control for LG and GW treatment


title: Day 17 RNA-seq in mES Rep2


title: Day 16 GW3965 RNA-seq in mES Rep2


title: 24h 1microM ATRA RNA-seq in mES Rep2

value: 24h ATRA 1mM


title: 24h 1microM ATRA RNA-seq in mES Rep1


title: 0h RNA-seq in mES Rep2

description: control for ATRA treatment


title: Day 17 GW3965 RNA-seq in mES Rep2


title: 0h RNA-seq in mES Rep1

description: control for ATRA treatment


title: E85H_Mark3

data_processing:
Library strategy: 4C-Seq
De-multiplexing, mapping and 4C-seq were done through HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
Reads were mapped to the mm9 (NCBIM37) Mouse genome assembly with bowtie version 0.12.7 (Langmead et al, 2009 Genome Biology) with parameters -n 2 --best -strata -m 5.
Mapped reads were translated to restriction fragments using the HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
4C figures were made using a running mean algorithm with a window size of eleven fragments.
Genome_build: mm9
Supplementary_files_format_and_content: Signal/restriction fragment (bedGraph) and locally normalized, smoothed (bedGraph), one per viewpoint, per cell type.

source_name: E8.5 head

extract_protocol:
Multiplexed samples were sequenced on the Illumina Genome HiSeq 2500 system using 100 bp single-end reads according to the manufacturer’s specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline of the BBCF HTSstation (David et al. 2014, Plos ONE and available at http://htsstation.epfl.ch). Samples were mapped to the ENSEMBL Mouse assembly NCBIM37 (mm9).
Illumina adapters (single end reads) were included in the inverse 4C PCR primers used for 4C-seq library preparation
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. Tissue sample were further made single cell by collagenase treatment and applying a cell-strainer cap (BD Biosciences). Chromatin was crosslinked in the presence of 2% formaldehyde.
PCR amplification using primers with included adapter sequences (for Illumina HiSeq2500)

treatment_protocol:
4C–seq libraries were constructed as described before (Matelot and Noordermeer, Methods in Molecular Biology 2016). 4C-seq libraries were prepared from 250 micro-dissected E8.5 embryonal heads. The primary restriction enzyme used was DpnII (New England Biolabs, R0125L) and the secondary restriction enzyme was NlaIII (New England Biolabs, R0543M). For each viewpoint 1400 ng of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Matelot and Noordermeer, Methods in Molecular Biology 2016). Cross-linked chromatin was digested with DpnII, ligated under diluted conditions, cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions.

tag: strain

value: head

tag: genotype


title: E85H_Krox20DS

value: WT


title: L_3

city: Grenoble

first: Christel

last: Carles

state: Auvergne/Rhone Alpes

address: 17 rue des Martyrs, bât. C2

department: Laboratoire Physiologie Cellulaire Végétale (LPCV)

institute: University Grenoble Alpes

data_processing:
Base-calling performed with Casava 1.8.2
Reads were aligned using bowtie2, 2.1.0
Mapping of reads to tair10 genome and determination of differentially expressed genes was performed using TopHat 2.0.10 and Cufflinks 2.1.1 with default parameters as described in Trapnell et al., (2012).
Genome_build: TAIR10
Supplementary_files_format_and_content: Tab-delimited text file containing normalized expression values in each tissue/ time point (denoted as Fragments Per Kilobase Of Exon Per Million Fragments Mapped (FPKM)).

source_name: rosette leaf

growth_protocol: All employed lines were in Landsberg errecta (Ler) ecotype background. ap1 and cal double mutants (ap1-1 cal-1) (Bowman et al., 1993) and 35S::AP1:GR ap1-1 cal-1 (Wellmer et al., 2006) were described previously. After 2-3 days of stratification at 4°C, seeds were germinated on Murashige and Skoog medium supplemented with 0.3 % sucrose, in growth cabinets (Percival) providing a controlled environment in long days (LD) of 16 hours light at 18°C and 8 hours darkness at 16°C. At about ten days after germination, plants were transferred to a 1:1 mixture of soil/vermiculite.

extract_protocol:
Total RNA was isolated with the RNeasy Plant Mini Kit (Qiagen) and 8 µg of RNA were DNase treated with DNA-freeTM Kit (Ambion).
Library preparation was performed using the Truseq stranded mRNA sample preparation kit (Illumina, ref.RS-122-2101 ) according to the manufacturer instructions. Briefly, polyadenylated RNAs were purified using oligo-d(T) magnetic beads, fragmented, and reverse transcribed using random hexamers, Super Script II (Life Technologies, ref. 18064-014) and Actinomycin D. During the second strand generation step, dUTP substituted dTTP. This prevented the second strand to be used as a matrix during the final PCR amplification. Double-stranded cDNAs were adenylated at their 3' termini before ligation was performed using Illumia's indexed adapters. Ligated cDNAs were amplified following 15 PCR cycles and PCR products were purified using AMPure XP Beads (Beckman Coulter Genomics, ref.A63881). Libraries were validated using a DNA1000 chip (Agilent, ref. 5067-1504) on a Agilent Bioanalyzer and quantified using the KAPA Library quantification kit (Clinisciences, ref. KK4824). Equimolar amounts of 4 libraries were pooled, diluted to 10 nM, denatured, and diluted again to 7 pM. 100 µl of the diluted pool were hybridized on one lane of an Illumina's Flow Cell. Clustering and 50 cycles sequencing were performed according to Illumina's instructions on a Hiseq2000.

value: wild type (Ler)

value: rosette leaves of flowering Arabidosis plants were harvested

tag: developmental stage

value: L


title: t0_2

source_name: inflorescence

value: ap1 cal 35S::AP1-GR

value: apices (only the cauliflower structures of the main inflorescence were harvested. Leaf and pedicel tissue contamination was minimized by dissection)


title: t2_2

treatment_protocol: 2 days after dexamethason treatment: plants were treated with induction solution (10 µM Dexamethason, 0.015 % Silvet) by pipetting the solution directly onto the cauliflower structures. This process war repeated once after 30 min.


title: I_2

value: inflorescences (whole main inflorescences of wild type plants were harvested. Flowers older than stage 12 were removed)

value: I


title: t2_1


title: t0_3


title: t0_1


title: t2_3


title: E85H_Krox20


title: E85H_ElemC


title: E85H_ElemB


title: I_3


title: GlcNAc ChIP-seq on GalNAz fed sxc null pupae

city: Burnaby

first: David

last: Vocadlo

state: British Columbia

address: 8888 University Drive

department: Chemistry

country: Canada

institute: Simon Fraser University

description: See Sinclair et al. (2009) for details

data_processing:
ChIP-seq reads were aligned to the mm9 genome assembly using bwa-mem or bwa-bwasw algorithm version 0.7.5a-r405.
Bam-format alignment files were generated with Samtools version 0.1.19-96b5f2294a.
Multiple runs were merged with Samtools.
Duplicate read-pairs were removed with Samtools.
BDG files generated with macs version1.4
Genome_build: dm3

organism: Drosophila melanogaster

source_name: GalNAz-fed-sxc-null-white-pre-pupae

extract_protocol:
Pools of ~25 pupae were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These pupae pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min). Before each enrichment of O-GlcNAz modified nuclear proteins, 500 µL solution (1% SDS/PBS) consisting of 800-1,000 µg nuclear protein was mixed with 50 µL Streptavidin-agarose slurry (Sigma), incubated for 1 h at 4°C for preclearing. The supernatant was transferred to a new tube and reacted with 200 µM Biotin-azo-phosphine for overnight at room temperature. Unreacted probe was removed by chloroform/methanol precipitation. Air-dried protein pellets were resuspended in 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0) and enriched by Streptavidin-agarose slurry (Sigma) for a couple of hours at 4 °C with gentle rocking. The beads were sequentially washed trice with 5-10 volumes of 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0), PBS, and 1% SDS/PBS. Centrifugation of the beads between washing steps was carried out (7,500 rpm, 2 min). Bound proteins were cleaved from the beads by treating with one beads volume of elution buffer (100 mM Na2S2O4 in 1% SDS/PBS) for 30 min trice. Collect and combine the eluants. Removal of the majority of Na2S2O4 from the eluants was achieved by precipitating them with chloroform/methanol method; 10% of each eluted sample was analyzed by Immunoblot using Odyssey (LI-COR Biosciences).
The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).

treatment_protocol: Crosses were grown at 25°C on cornmeal-molasses-yeast medium supplemented with the mold inhibitors tegosept and propionic acid. Ac4GalNAz media was made by adding Ac4GalNAz at 55°C to a final concentration of 100µM. Parents mated on this food and the progeny grew up on it since birth. Approximately 25 white pre-pupae were harvested for ChIP-seq library prep which was accomplished in a similar process as Ac4GalNAz fed S2 cells.

value: pupae

tag: chip antibody/selection

value: GalNAz feeding, Staudinger ligation, and streptavidin purification


title: L_2


title: I_1


title: L_1


title: GlcNAc ChIP-seq on GalNAz fed wt pupae

description: See Sinclair et al. (2009) for details

source_name: galNAz-fed-wt-white-pre-pupae

value: Oregon R


title: input genomic DNA from S2 cells

growth_protocol: Drosophila melanogaster Schneider S2 cells were cultured in Sf-900 II SFM medium (Invitrogen) supplemented 100 U/ml Penicillin and 100 mg/ml Streptomycin at 25°C. Cells were passaged at 1:4 ratio every two days to keep logarithmic growth.

extract_protocol:
~2x107 cells were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These cell pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min).
The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).

treatment_protocol: Cells were not treated.

value: Schneider's Drosophila Line 2 (SL2) cells

tag: cell line

tag: passages

value: Genomic DNA (input)


title: E85H_ElemA

tag: age


title: GlcNAc ChIP-seq on S2 cells using GalT purification

extract_protocol:
~2x107 cells were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These cell pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min). To label O-GlcNAcylated proteins with Ac4GalNAz, the Click-iT O-GlcNAc Enzymatic Labeling System was used (Invitrogen). Briefly, Gal-T1Y289L was incubated with proteins in labeling buffer (containing 20 mM HEPES, pH 7.9; 5 mM NaCl; 2% NP-40; 5.5 mM MnC2; 25 µM UDP-GalNAz), according to manufacturer's recommendations. Reaction was performed at 4°C under gentle agitation for 24 h. All reagents were provided in the kit. Once labeling achieved, proteins were chloroform/methanol precipitated. Note that the volume of each reagent was adjusted for higher proteins quantity, i.e., when 500 µg of proteins were labeled.
The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).

value: Purification with UDP-GalNAz and GalT labeling followed by click chemistry and streptavidin purification


title: E95repl_Nrbf2

source_name: E9.5 full embryo - replicate

treatment_protocol:
4C–seq libraries were constructed as described before (Matelot and Noordermeer, Methods in Molecular Biology 2016). 4C-seq libraries were prepared from 35 E9.5 embryos. The primary restriction enzyme used was DpnII (New England Biolabs, R0125L) and the secondary restriction enzyme was NlaIII (New England Biolabs, R0543M). For each viewpoint 1000 ng of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Matelot and Noordermeer, Methods in Molecular Biology 2016). Cross-linked chromatin was digested with DpnII, ligated under diluted conditions, cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions.

value: full embryo - replicate


title: t2_input_rep_2_for_K27

data_processing:
Base-calling performed with Casava 1.8.2
reads were aligned using bwa 0.6.2-r126
files were converted into bam formate using samtools 0.1.19+
duplicated reads were removed using Picard-tools 1.93
bed files were generated using bedtools v2.17.0-97-ge80528b
H3K27me3 and H3K4me3 positive islands were determined using SICER V1.1 with window size 200 bp, gap size 400 bp and effective genome size of 90 % , a FDR-cutt-off of 0.0001 was employed
Genome_build: TAIR10
Supplementary_files_format_and_content: Significantly enriched islands (determined with input as control), csv-files, containing Chr, start, stop, ChIP_island_read_count, CONTROL_island_read_count, p_value,  fold_change, FDR_threshold

extract_protocol:
Formaldehyde cross-linking and extraction of nuclei was performed as described in Carles and Fletcher (2009) with the following modifications: infiltration time was prolongated (6 min and 24 min), filtration of the extract after grinding was performed through 75 µm and 50 µm nylon meshes and sonication was done for 13x 30 sec on, 1 min off, position H (this longer sonication was only performed for biological replicate 2). Immunoprecipitation reactions were set up as described in Carles and Fletcher (2009) except that 15 µg of cauliflower and 10 µg of wild type chromatin was used and the chromatin was incubated with 5 µl of the antibody alone for 5 hours prior to addition of Protein A Agarose beads without salmon sperm DNA (Millipore). Washing of the ChIP reaction was performed using binding/wash buffer as described in Carles and Fletcher (2009) but for four times 10 min at 4°C and once 10 min at room temperature (RT).   Elution and reverse cross-linking was performed as described in Kaufmann et al. (2010) except that the elution was done by vortex mixing for 1 min at RT and input samples (1/3 of the chromatin used for one ChIP sample) were brought to the same volume by adding elution buffer and Tris pH9 in the same ratio as to the ChIP samples.  Reverse cross-linked samples were purified using the MinElute Reaction Cleanup Kit (Quiagen) and DNA was eluted in 30 µl Elution buffer.  For biological replicate 2, three technical replicates of ChIP on the same nuclear extract were performed and pooled on one MinElute column.
Libraries from ChIP samples of Replicate 1 were prepared and sequenced on a GAIIx (Illumina) sequencer as described previously (Yant et al., 2010; Immink et al., 2012). For biological replicate 2, ChIP-seq library preparation was performed using the Truseq ChIP sample preparation kit (Illumina, ref. IP-202-1012) according to the manufacturer's instructions except that PCR amplification was performed prior to size selection. Briefly, sonicated chromatin was repaired prior to 3' ends adenylation. Illumina's indexed adapters were ligated to the adenylated double-stranded DNA fragments. A 18-cycles PCR was performed on the ligated DNA using Illumina's PCR primers. The libraries were then size-selected between 300 and 700 bp on a 2% agarose gel. Each library was validated using high sensitivity chip (Agilent, ref. 5067-4626) on an Agilent Bioanalyzer and quantified using the Kapa library quantification kit (Clinisciences, ref. KK4824). Equimolar amounts of 4 libraries were pooled, diluted to 10 nM, denatured, and diluted again to 7 pM. 100 µl of the diluted pool were hybridized on one lane of an Illumina's Flow Cell. Clustering and 50 cycles sequencing were performed according to Illumina's instructions on a Hiseq2000. Libraries were multiplexed by three (H3K4me3 samples) or four and sequenced on a HiSeq2000 (Illumina) sequencer, yielding 50 bp single-end reads.
All inputs were sequenced with the HiSeq2000.

treatment_protocol: 2 days after dexamethason treatment : plants were treated with induction solution (10 µM Dexamethason, 0.015 % Silvet) by pipetting the solution directly onto the cauliflower structures. This process war repeated once after 30 min.

tag: chip antibody


title: I_input_rep_2_for_K27


title: t2_input_rep_2_for_K4