title: poolD_mouse1


title: Epi_Dnmt3b_6_Perturbation-RNA-Seq

status: Public on Sep 18 2017

type: SRA

city: Boston

first: Jiantao

last: Shi

address: 3 Blackfan Circle

country: USA

institute: dana farber cancer institute

library_source: transcriptomic

data_processing:
Alignment was performed using TopHat against mouse genome assembly mm10 with default settings.
Kallisto was used for quantification of transcript-level expression, which was reported as TPM.
Genome_build: mm10
Supplementary_files_format_and_content: text file contains transcript-level expression, represented as TPM.

molecule: total RNA

organism: Mus musculus

source_name: Epiblast with Dnmt3b knockout

extract_protocol:
Genomic DNA and mRNA purifications from low input samples were performed as described by Maculay et al. 2015 with modifications. Briefly, the cells were mixed with 15 µl of RLT plus buffer (QIAGen) containing 1 U/µl of RNase inhibitor (SUPERase·In, ThermoFisher Scientific), 1% ß-mercaptoethanol (Sigma-Aldrich), and were then transferred to one well in a 96-well DNA LoBind plate (Eppendorf). After adding 10 µl of M-280 streptavidin bead-conjugated RT primer to each sample, the reaction was incubated at 72˚C for 3 min in a thermocycler followed by incubation at room temperature for 25 min with gentle rotation. The genomic DNA and mRNA were separated in a DynaMag™-96 Side Magnet (ThermoFisher Scientific).
Bead-tagged mRNA was subjected to reverse transcription as described previously in Maculay et. al 2015 and the genomic DNA in the supernatant was transferred to a fresh 96-well DNA LoBind plate. After reverse transcription, the cDNA was PCR amplified and RNA-seq library was generated according to the Smart-seq 2 protocol. Indexed RNA-seq libraries were pooled and sequenced in an Illumina Hiseq2500 sequencer.  Genomic DNA was isolated utilizing 1x Agencourt AMPure beads (Beckman Coulter) and was eluted to 15 µl of low TE buffer. RRBS library was generated as reported previously in Gu et. al 2013 with modifications. We utilized the CutSmart buffer (New England Biolabs) for all three enzymatic reactions including MspI digestion, end-repair/A-tailing and T4 DNA ligation. To minimize DNA loss, DNA purification step was eliminated after each enzymatic reaction. Briefly, the genomic DNA was digested by 16 units of MspI (New England Biolabs) for 80 min at 37˚C, and followed by heat inactivation at 65˚C for 15 min. The digested DNA fragments were end-repaired and A-tailed by adding 4 units of Klenow exo- (New England Biolabs), 0.03 mM dCTP, 0.03 mM dGTP and 0.3 mM dATP; ant the reaction was carried out at 30˚C for 25 min; 37˚C for 25 min followed by incubation at 70 ˚C for 10 min to inactive the enzyme. We then ligated the A-tailed DNA fragments with indexed adapters overnight at 16 ˚C by adding 2,000 units of T4 DNA ligase and 0.75 mM ATP and 7 nM of the adapters. The T4 ligase was heat-inactivated at 65 ˚C for 15 min before pooling libraries together. To remove adapter dimers, the library pool was cleaned up using 1.8X AMPure beads and the adapter-tagged DNA fragments were eluted to 30 µl of low TE buffer. The bisulfite conversion of the adapter-tagged DNA fragments ware conducted using a QIAGen EpiTect Fast Bisulfite Conversion Kit following the manufacturer’s instructions with a minor modification. We extended the bisulfite conversion time from 2 cycles of 10 min to 2 cycles of 20 min to achieve bisulfite conversion rates >99%. The bisulfite converted DNA fragments were PCR amplified according to the following thermocycler settings: 98 ˚C for 45 s, 6 cycles of 98 ˚C for 20 s, 58 ˚C for 30 s, 72 ˚C for 1 min and then 8-10 cycles of 98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 1 min followed by a final extension cycle of 5 min at 72 ˚C. The PCR amplified library DNA was cleaned up using 1.3X AMPure beads and the RRBS libraries were paired-end sequenced for 2x100 cycles.  Only instances where the matched pool of Epiblast and ExE from a given replicate both had over 1 million CpGs covered at ≥5x were included for downstream analysis.  For each sample, 10 µl of M-280 streptavidin beads (ThermoFisher Scientific) were prepared per the manufacturer’s recommendations. Specifically, after washing with Solution A (0.1 N NaOH, 0.05 M NaCl) and B (0.1 M NaCl) sequentially, the beads were resuspended in 10 µl of 2x Washing and Binding buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl) and then mixed with an equal volume of 2 µM of RT primer (Maculay et al. 2015). The mixture was incubated for 15 min at room temperature with gentle rotation. The bead-bound RT primer was collected in a magnate and was subsequently resuspended in 10 µl of binding buffer (10 mM Tris-HCl (PH 8.0), 167 mM NaCl, 0.05% Tween-20).

treatment_protocol: Outgrowth  experiments:  To generate controlled outgrowth data, ICM were immunosurgically isolated from BDF1x129S1/SvImJ strain blastocysts at 96hpf.  Briefly, oocytes were isolated by hormone priming from B6D2F1 females 12-14 hours after administration of hCG and fertilized by Intracytoplasmic Sperm Injection using piezo actuated injection of 129S1/SvIMJ strain sperm.  At 96 hrs post-fertilization, blastocysts were stripped of their zona pellucida by brief incubation in Acid Tyrode’s solution and incubated for 30 minutes in 1:10 diluted whole mouse antisera (Sigma) in CO2 equilibrated KSOM, followed by destruction of the trophectoderm by culture in 1:10 diluted Guinea Pig Complement Sera (Sigma).  After 15 minutes at 37ºC, the ICM separates from the complement-lysed TE and could be cleanly isolated by brief pulsing through a narrow glass capillary.  ICM were isolated in batches of ~12 per drop. Once isolated, ICM were then plated into basal N2/B27 media supplemented with 1000 U/mL LIF (made in house) and one of the following conditions; ‘2i’ supplemented with 1 mM PD0325901 and 3 mM CHIR99021 (Reagents Direct); ‘PD’ supplemented with  1 uM PD0325901 and 10 ng/mL BMP4 to promote outgrowth expansion (Peprotech), ‘FGF+CHIR’ supplemented with 25ng/mL mouse recombinant FGF4 (R and D) and 3 mM CHIR99021; and ‘FGF’ supplemented with 25ng/mL FGF4 only.  FGF4 was selected because it is the most highly expressed FGF family member in the preimplantation embryo and we sought to direct specific remethylation changes as is observed in vivo.  ICM were plated in gelatin treated tissue culture dishes plated with irradiated CF-1 strain embryonic fibroblasts to promote attachment.  The primary outgrowth from the ICM, characterized as a centrally expanding, three-dimensional mass, was isolated after 4 days of culture.  In all cases but the 2i condition, an outer layer of differentiated cells was apparent and removed using an identical strategy to removal of the extraembryonic endoderm from E6.5 samples described above.  However, under the FGF+CHIR condition, the ‘outer layer’ was often of the same size or larger than the internal outgrowth and only became defined during the latter portion of culture.  As such, we collected both interior and exterior portions as they could clearly be distinguished as mutually ICM-derived.  After incubation and either isolation or removal of external cells, outgrowths were serially washed through several KSOM drops under mineral oil before being snap frozen in minimal volume for RRBS and RNA-seq profiling.  Generation of KO embryos by CRISPR/Cas9 and zygotic injection:  Zygotic injection was performed essentially as described (Wang et. al, 2013 Cell) To improve the efficiency with which null alleles were generated, three separate guide RNA sequences were designed per target, prioritizing highly scored protospacer sequences with no high scoring off target sites using the CHOPCHOP web tool and at as 5’ most a coding exon as possible given these constraints.  Protospacer sequences were input into the following oligonucleotide primer pair and used to amplify off of the pX300 plasmid (Addgene): Forward primer, AGTCAGTTAATACGACTCACTATAGN19GTTTTAGAGCTAGAAATAGCAAG; Reverse primer, AAAAAAAGCACCGACTCGGTGCCAC.  Protospacer sequences that did not begin with a G to initiate T7 transcription were inserted and an additional 5’ G was added.  200 ng of gel purified, T7 promoter containing sgRNA templates were used to generate gRNAs by in vitro transcription using the MEGAshortscript™ T7 transcription kit (Thermo Fisher), followed by purification with phenol:chloroform and ethanol precipitation.  Translation competent spCas9 RNA was in vitro transcribed off of a similarly designed, T7 promoter driven template amplified off of the pX300 plasmid using the mMessage mMachine™ T7 Ultra kit (Thermo Fisher) and purified using the RNA Clean and Concentrator Kit (Zymo Research).  RNA was resuspended in an injection buffer comprised of 5 mM Tris-HCl, 0.1 mM EDTA, pH=7.4.  Zygotes were isolated from hormone primed B6D2F1females mated with B6D2F1 males as described above.  Shortly after the formation of visible pronuclei (Pronuclear Stage 3), zygotes were cytoplasmically injected with 100 ng/ul of all three targeted sgRNAs pooled 1:1:1 and 200 ng/ul Cas9 mRNA.  At E3.5, cavitated blastocyts were transferred in clutches of 10-15 into one uterine horn of pseudopregnant CD-1 strain mice (Charles River) that had been mated with vasectomized male Swiss-Weber strain mice (Taconic) two days prior.  To account for the ~1 day offset in developmental progression that results from uterine transfer, appropriately E6.5 stage conceptuses were isolated four days after uterine transfer and epiblast and extraembryonic ectoderm tissue were isolated.  Each replicate consisted of ≥4 embryos and all experimental series include replicates generated from at least two rounds of zygotic injection.  Care was taken to ensure epiblast and extraembryonic ectoderm tissue from matched embryos were included for each replicate set and RRBS data where both fractions did not cover >1 million CpGs at ≥5x coverage each were excluded from further analysis.

tag: developmental stage

value: Epiblast

tag: ko


title: poolD_input4

city: London

first: M Joaquina

last: Delas

address: 1 Midland Road

department: Briscoe Lab

country: United Kingdom

institute: The Francis Crick Institute

library_source: genomic

data_processing:
Reads were extracted and mapped to the shRNAs of the corresponding pool using bowtie (allowing 0 mismatches). Fasta file for the shRNA also included
To analyze the depletion between the final timepoint and input, DESeq was used with Fit type “local”
Genome_build: NA
Supplementary_files_format_and_content: Screen_DESeq_allPools.xls contains the results of DESeq analysis, mjdelasv_GEO_AMLscreen-shRNAs.fasta contains the shRNAs names and sequence.

molecule: genomic DNA

organism: synthetic construct

source_name: RN2 cells (MLL-AF9/NRASG12D)

growth_protocol: Pools of 50 shRNA vectors were used to produce virus and transduce AML cells at a low multiplicity of infection to minimize double infections. Cells were treated with 500 μg ml-1 G-418 (Roche Applied Science) from two days after infection until fully selected.

extract_protocol:
Library preparation was performed as previously described (Knott (2014) Mol Cell).
In brief, genomic DNA was extracted from the pre-injection pool and the bone marrow cell suspensions using the QIAamp Blood DNA Maxi Kit (Qiagen). For each sample, shRNA hairpins were extracted from genomic DNA in 96 separate 25-cycle PCR reactions where 2 mg of input DNA was included in each reaction.
Following this initial PCR, Illumina adapters were added via PCR, and samples were processed on the Illumina MiSeq platform.

treatment_protocol: 1 x 106 fully selected AML cells were injected in the tail vein of sublethally irradiated (4.5 Gy, 24h before injection) B6.SJL (CD45.1) female mice 6-8 weeks of age. For shRNA induction, animals were treated with doxycycline in the food (625 mg kg-1, Harlan Laboratories). Leukemic mice were euthanized 14 days after transplantation, at terminal disease stage, by CO2.

tag: shRNA pool

value: poolD

tag: timepoint

value: Injection pool


title: poolD_input2


title: poolD_input3


title: poolC_mouse4

value: poolC

value: bone marrow day 14


title: poolC_mouse2


title: poolD_input1


title: Epi_Dnmt3b_4_Perturbation-RNA-Seq


title: Epi_Dnmt3b_3_Perturbation-RNA-Seq


title: poolC_mouse3


title: poolC_mouse1


title: poolC_input4


title: Epi_Dnmt3b_1_Perturbation-RNA-Seq


title: Epi_Dnmt3b_2_Perturbation-RNA-Seq


title: poolD_mouse5


title: poolC_input2


title: Epi_Dnmt3a_6_Perturbation-RNA-Seq

source_name: Epiblast with Dnmt3a knockout


title: poolC_input1


title: poolB_mouse2

value: poolB


title: poolB_mouse4


title: poolB_mouse1


title: Epi_Dnmt3b_5_Perturbation-RNA-Seq


title: poolC_input3


title: poolB_input3


title: Epi_Dnmt3a_5_Perturbation-RNA-Seq


title: poolB_mouse3


title: poolB_input4


title: poolB_input2


title: Epi_Dnmt3a_1_Perturbation-RNA-Seq


title: poolA_mouse4

value: poolA


title: poolA_mouse3


title: poolA_mouse2


title: poolA_mouse1


title: Epi_Dnmt1_4_Perturbation-RNA-Seq

source_name: Epiblast with Dnmt1 knockout


title: Epi_Dnmt1_3_Perturbation-RNA-Seq


title: poolA_input4


title: ES2-Liquiritigenin-2

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

description: ES2 cells trated with liquiritigenin

data_processing:
Illumina bcl2fastq v2.17.1.14 software used for base-calling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19 whole genome using TopHat v2.0.8b
gene count measurements were obtained using HTSeq-count
Genome_build: hg19
Supplementary_files_format_and_content: The processed data files are in tabular format.  The first column contains the gene symbols and the second column contain the read counts of the gene using htseq-count

organism: Homo sapiens

source_name: ES2 ovarian cancer cell line

growth_protocol: ES2 were cultured in phenol red–free RPMI-1640 medium containing 5% dextran-charcoal treated FBS

extract_protocol:
ES2 cells were treated with either vehicle or liquiritigenin for 24 h, and total RNA was isolated using RNAesy mini kit (Qiagen) according to the manufacturer’s instructions. The purity of isolated RNA was determined using Agilent 2100 BioAnalyzer. Illumina TruSeq RNA Sample preparation was performed following manufacturer's protocol, and the samples were run on an Illumina HiSeq 2000 in duplicates.
Total RNA samples about 1-2 micrograms were used for sequencing library preparation according to Illumina TruSeq Total RNA Sample Preparation Guide (Illumina Cat. #: RS-122-2201). Each library was bar-coded and then pooled for cluster generation and sequencing run with 100bp single-end (SE) sequencing protocol.

treatment_protocol: Total RNA was isolated from ES2 cells that are trated with vehicle or liquiritigenin for 24 h were subjected to RNA-seq analysis

tag: treatment

value: Treatment


title: Epi_Dnmt1_2_Perturbation-RNA-Seq


title: Epi_Dnmt3a_4_Perturbation-RNA-Seq


title: ES2-Liquiritigenin-1

description: ES2 cells trated with liquiritigenin


title: ES2-control-2

description: ES2 cells trated with vehicle

value: Control


title: ES2-control-1

description: ES2 cells trated with vehicle


title: Epi_Dnmt3a_2_Perturbation-RNA-Seq


title: poolA_input2


title: Epi_Dnmt1_1_Perturbation-RNA-Seq


title: poolB_input1


title: Gal4

city: Aurora

first: Jay

middle: R.

last: Hesselberth

state: CO

department: Biochemistry and Molecular Gentetics

institute: University of Colorado School of Medicine

data_processing:
Aligned to sacCer1 using Bowtie -m 1
Created bedfile
Normalized bedfile to # reads aligned (Reads per million, RPM)
Created bigWig file
Genome_build: sacCer1
Supplementary_files_format_and_content: Normalized bigWig containing genome position and # reads for Calling Cards Ty5 Insertion signal (measures location of DNA replication factor or transcription factor binding)

organism: Saccharomyces cerevisiae

source_name: Budding yeast

growth_protocol: S. cerevisiae grown on glucose containing media plates at 30C, transferred to galactose containing media plates at 22C to induce transposition and then grown at 30C on selective plates to select for cells containing integrated Ty5

extract_protocol:
Yeast were spheroplasted to obtain high molecular weight DNA treated with RNAse, DNA was digested using three 4bp restriction enzymes
DNA libraries were created using inverse PCR amplification of ligated circular DNA with primers containing Truseq adapters, and single read sequencing was performed on the Illumina HiSeq 2000 platforms using 50bp reads
Four DNA Truseq primers were used containing unique 6bp bar codes that allowed each construct to be marked with one of the unique barcodes.  Each PCR reaction was diluted to 10nM and then were pooled together and submitted directly for sequencing.  Calling Cards plasmid design required a secondary read 2 primer to be added to the reaction.

tag: strain background


title: 2i_4_Perturbation-RNA-Seq

source_name: ICM outgrowth under 2i condition

value: ICM outgrowth

value: PD + CHIR


title: poolA_input1


title: Ty5


title: cdc7kdΔcterm


title: 2i_3_Perturbation-RNA-Seq


title: Epi_Dnmt1_5_Perturbation-RNA-Seq


title: 2i_2_Perturbation-RNA-Seq


title: cdc7kd


title: GS9

city: Beijing

first: huanwei

last: huang

address: 7 Science Park Rd., ZGC life science park

country: China

institute: NIBS

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm9 whole genome using tophat v2.1.0 with default parameters.
Raw reads number of genes was calculated by HTSeq. cufflinks v2.2.1 was run for relative quantitation of genes expression and differential expression analysis.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.

growth_protocol: ES cells and iPS cells were maintained in ES cell media containing 15% FBS and 1,000 Uml-1 of LIF.MEF and APCs were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin.

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen) from different cell lines. Libraries were constructed with NEBNext DNA Library Prep Master Mix Set for Illumina (New England Biolabs), and PCR products were purified by AMPure XP beads (Beckman). RNA library was quantified by Qubit 1.0((Invitrogen) and analyzed with agilent 2100 bioanalyzer (Agilent) for size distribution, sequenced with Illumina Hiseq-2500 in single mode (1x50nt).
libraries were prepared for sequencing using NEBNext Ultra DNA Library Preparation Kit for Illumina

tag: strain

tag: cell type

value: APC-derived induced pluripotent stem cells

tag: passage


title: GS32


title: 2i_1_Perturbation-RNA-Seq


title: GS4


title: AI10


title: AI9


title: AI7


title: poolA_input3


title: MOLM13 METTL3 shRNA #12 Ribo-seq rep 3

city: New York

first: Brian

middle: Frederick

last: Pickering

state: New York

address: 1300 York Ave

department: Pharmacology

institute: Weill Cornell Medicine

description:
ribosome-depleted RNA
rpf.counts.xls
m3kd.12.3.rpf

data_processing:
Library strategy: Ribo-Seq
miCLIP : Low-quality bases, reads with more than two ambiguous base calls, and adapter sequence were removed using FLEXBAR tool (--max-uncalled 2 --min-read-length 15 --pre-trim-phred 30, 3’ adapter: AGATCGGAAGAGCGGTTCAG). Then, PCR-duplicated reads of identical sequences were removed using the pyDuplicateRemover.py script of the pyCRAC tool suite (version 1.2.2.3). Unique reads were mapped with Novoalign (v3.02.12) using hg19 as reference genome. Further analysis including the identification of CITS was performed as previously described (see GSE63753). A custom perl script was then used to filter the identified putative m6A residues from the CITS file. This script filters C to T transitions based on the number of transitions (m ≥ 2) and the transition frequency (1% ≥ m/k ≤ 50%) at any given coordinate. Additionally, we filtered for those called sites that have a DRACH sequence.
Ribo-seq : Adapters were trimmed using FLEXBAR tool (--min-read-length 12, --pre-trim-phred 32, 3’ adaptor: AGATCGGAAGAGCACACGTCT).  Ribosomal RNA reads were removed using STAR aligner22.  Gene count tables were generated using STAR aligned to hg19 transcriptome and normalized using DESeq223
Genome_build: hg19
Supplementary_files_format_and_content: bed files contain miCLIP sites from biological triplicates, counts tables are normalized read counts per gene for RNA-Seq and Ribo-Seq used for GSEA enrichment and cumulative distribution plots.

source_name: MOLM13 ribosome-depleted RNA

growth_protocol: Cells were grown as per ATCC guidelines.

extract_protocol:
miCLIP RNA extract protocol: Total RNA was extracted from MOLM13 cells with Trizol (n=3 biological replicates).  Poly(A) mRNA was then isolated using two rounds of selection with oligo(dT) magnetic beads (New England Biolabs).  30 ug of poly(A) RNA was used for each replicate in this study.
miCLIP library construction protocol: miCLIP libraries were prepared as previously described (PMID: 28349454).
RNA-seq and Ribo-seq library construction protocol: MOLM13 cells were transduced with control shRNA or two independent shRNAs targeting METTL3.  Four days post-transduction 3x10^6 MOLM13 cells were washed twice with ice-cold PBS pelleted by centrifugation.  After pelleting, cells were resuspended in 300 μl lysis buffer (25 mM HEPES-KOH pH 7.2, 200 mM KOAc, 1% NP-40, 10 mM MgCl2, 4 mM CaCl2) for 5 minutes on ice then centrifuged at 8000 x g for 5 minutes.  100 μl of lysate was mixed with TRIzol and saved as input.  To generate ribosome protected fragments the lysates were first mixed with 200 μl DEPC-H2O then incubated with 10 μg/ml micrococcal nuclease for 30 min at 37°C.  RNA was extracted with TRIzol LS.  T4 PNK was used to generate RNA fragments with a 5’ phosphate and 3’ hydroxyl.  The RNA was then run on a 15% 8M urea TBE gel, stained with SYBR Gold, and a gel fragment between 27-35 nucleotides corresponding to ribosome protected RNA was excised.  RNA was eluted overnight in 400 μl TE after crushing the gel fragment.  RNA was ethanol precipitated and resuspended in 7 μl water.  To generate libraries from RNA protected fragments 3 μl of the RNA was used with the NEBnext small RNA library prep kit (NEB) according to the manufacturer’s instructions.  Due to the similarity in size between ligated and unligated adapters, prepared libraries (~155 nt) were gel purified. Ribosomal RNAs were removed from inputs using NEBNext rRNA Depletion Kit (NEB).  Input RNA libraries were generated using the NEBNext Ultra Directional RNA library prep kit for Illumina (NEB). Libraries were sequenced using an Illumina HiSeq 2500 platform with 50 nucleotide reads.

treatment_protocol: For RNA-Seq and Ribo-Seq experiments MOLM13 cells were transduced with lentivirus expressing control shRNA or two shRNAs targeting METTL3 (M3KD9 & M3KD12).  RNA was collected four days post-transduction.

value: Acute myeloid leukemia

tag: antibody name

tag: number of replicates

tag: barcodes

tag: shRNA

value: METTL3 shRNA #12


title: AI3


title: MOLM13 METTL3 shRNA #12 Ribo-seq rep 2

description:
ribosome-depleted RNA
rpf.counts.xls
m3kd.12.2.rpf


title: AN15

value: APC-derived nuclear transfer embryonic stem cells


title: MOLM13 METTL3 shRNA #9 Ribo-seq rep 3

description:
ribosome-depleted RNA
rpf.counts.xls
m3kd.9.3.rpf

value: METTL3 shRNA #9


title: MOLM13 METTL3 shRNA #9 Ribo-seq rep 2

description:
ribosome-depleted RNA
rpf.counts.xls
m3kd.9.2.rpf


title: AN9


title: AN7


title: MOLM13 METTL3 shRNA #9 Ribo-seq rep 1

description:
ribosome-depleted RNA
rpf.counts.xls
m3kd.9.1.rpf


title: AN5


title: MOLM13 Control shRNA Ribo-seq rep 3

description:
ribosome-depleted RNA
rpf.counts.xls
ctrl.3.rpf

value: Control shRNA


title: MOLM13 Control shRNA Ribo-seq rep 2

description:
ribosome-depleted RNA
rpf.counts.xls
ctrl.2.rpf


title: AN6


title: MOLM13 Control shRNA Ribo-seq rep 1

description:
ribosome-depleted RNA
rpf.counts.xls
ctrl.1.rpf


title: AN3


title: AN1


title: AN2


title: Hi-C ThymoDp(A)/p(A)

city: La Jolla

first: Kees

last: Murre

state: CALIFORNIA

address: 9500 gilman drive, NSB 5114A

department: Biology

institute: UCSD

description: used for Fig. 4

data_processing:
Reads were first trimmed from the 3’ end of sequences to GATC (MboI restriction enzyme site). Trimmed reads were aligned to mm9 with Bowtie software with parameters --chunkmbs 128 –mm -m1 --best --strata -p4 -S. The remainder of the analysis was performed using Homer. Only unique reads and paired end tags with more than 1.5x fragment length on the same chromosome separating them were retained. Sequence reads were checked for GC and nucleotide bias. HiC reads derived from wild-type DN2 and ThymoD-/- DN2 cells were compared to pro-B (GSM987818) and pre-proB/multipotent progenitors (GSM987816). Processed data files for these re-analyzed samples are available on the series record. Tag counts for all directories were normalized to the sample with lowest sequencing depth. For multipotent progenitors, pro-B and wild-type DN2 comparisons a map resolution of 50 kb was chosen to ensure that > 80% of loci were associated with at least 1000 genomic contacts. A superRes of 100 kb was used for comparison of wild-type DN2 and ThymoD-/- DN2 cells. A resolution of 40 kb (and 80 kb superRes) was chosen to ensure that greater than 80% of loci were associated with at least 1000 contacts. Hi-C heatmaps were normalized to output the ratio of observed to expected interactions by assuming each region has an equal chance of interacting with other regions in the genome and that regions are expected to interact depending on their linear distance along the chromosome. Hi-C heatmaps were visualized with Java Tree View. Circos diagrams were generated with Circos software. Only interactions with p-values <= 0.0001 are shown. Line thickness of Circos diagrams refers to p-values. Darker lines depict a lower p-values. In depth explanations of normalization, generation of Hi-C correlation matrices, principal component analysis (PCA) and finding significant interactions were performed as previously described (Lin, Y. C. et al. Nature immunology 13, 1196-1204, 2012).
genome build: mm9
processed data files format and content: the HiC matrix is matrix generated by Homer, and the values are logged ratio of observed to expected interactions.

source_name: in-vitro DN2 cells

growth_protocol: adult bone marrow single cell suspension were incubated with CD3e, B220, CD11b, GR1, TER119, CD117 and Flt3. Hematopoietic progenitor cells were collected through  depleting positive cells on Automacs. Selected cells were cultured on OP9-DL1 in 6 well plates with MEM containing 20% FCS, 2% PSG, FLT3L, recombinant IL-7 1-2 ng/ml.

extract_protocol:
Harvested cells were stained with lineage marker, CD25, CD117, ghost dye red 780. Cells were fixed with PBS containing 1% formaldehyde. Formaldehyde was quenched with 0.2 M glycine for 10 min. Lineage negative DN2 cells were sorted by FACS AriaII and washed with PBS. Fixed cells were stored at -80 degrees until use.
We used in-situ Hi-C to generate a genome-wide contact map essentially as described (Rao, S. S. et al. Cell 159, 1665-1680 2014). Five million cells each of wild-type DN2 and ThymoD-/- DN2 cells were generated using OP9-DL1 stromal cells. Nuclei were resuspended in 0.5% SDS diluted in a final concentration of 1X NEB Buffer 2. Each sequencing library was incubated with USER enzyme for 15 minutes at 37oC after step 56. Final libraries were submitted to paired-end sequencing of 100 bp length on an Illumina HiSeq 2500.

tag: genotype

value: ThymoDp(A)/p(A)

tag: chip antibody


title: Hi-C WT

description: used for Fig.4

value: WT


title: MOLM13 METTL3 shRNA #12 RNAseq rep1

description:
ribosome-depleted RNA
rnaseq.counts.xls
m3kd121

data_processing:
miCLIP : Low-quality bases, reads with more than two ambiguous base calls, and adapter sequence were removed using FLEXBAR tool (--max-uncalled 2 --min-read-length 15 --pre-trim-phred 30, 3’ adapter: AGATCGGAAGAGCGGTTCAG). Then, PCR-duplicated reads of identical sequences were removed using the pyDuplicateRemover.py script of the pyCRAC tool suite (version 1.2.2.3). Unique reads were mapped with Novoalign (v3.02.12) using hg19 as reference genome. Further analysis including the identification of CITS was performed as previously described (see GSE63753). A custom perl script was then used to filter the identified putative m6A residues from the CITS file. This script filters C to T transitions based on the number of transitions (m ≥ 2) and the transition frequency (1% ≥ m/k ≤ 50%) at any given coordinate. Additionally, we filtered for those called sites that have a DRACH sequence.
Ribo-seq : Adapters were trimmed using FLEXBAR tool (--min-read-length 12, --pre-trim-phred 32, 3’ adaptor: AGATCGGAAGAGCACACGTCT).  Ribosomal RNA reads were removed using STAR aligner22.  Gene count tables were generated using STAR aligned to hg19 transcriptome and normalized using DESeq223
Genome_build: hg19
Supplementary_files_format_and_content: bed files contain miCLIP sites from biological triplicates, counts tables are normalized read counts per gene for RNA-Seq and Ribo-Seq used for GSEA enrichment and cumulative distribution plots.


title: MOLM13 Control shRNA RNAseq rep 3

description:
ribosome-depleted RNA
rnaseq.counts.xls
m3kd93


title: MOLM13 METTL3 shRNA #12 RNAseq rep 2

description:
ribosome-depleted RNA
rnaseq.counts.xls
m3kd122


title: MOLM13 METTL3 shRNA #9 RNAseq rep 3

description:
ribosome-depleted RNA
rnaseq.counts.xls
ctrl3


title: MOLM13 Control shRNA RNAseq rep 2

description:
ribosome-depleted RNA
rnaseq.counts.xls
m3kd92


title: RNAseq DN2 ThymoDp(A)/p(A) 2

description: used for Fig. 3

data_processing:
RNAseq data were examined using Omics Pipe applying the RNA-seq count-based differential expression analysis pipeline. Quality control of the raw fastq files was performed using FastQC (Babraham Bioinformatics). Sequencing reads were aligned to the mouse genome (mm10) applying STAR aligner. Read quantification (exons) was performed using htseq-count with UCSC RefSeq annotation. The R BioConductor package DESeq2 was used to calculate size factors in order to normalize library sizes across replicates and calculate means and variances based on a negative binomial distribution model in order to detect differentially expressed genes based on adjusted p-values of < 0.05. Functional enrichment of the differentially expressed genes was done using ToppGene Suite, WebGestalt and Metascape.
genome build: mm10
processed data files format and content: The RNA-reads table are raw read-count generated by htseq-count.

extract_protocol:
Harvested cells were stained with lineage marker, CD25, CD117, ghost dye red 780. Lineage negative DN2 cells were sorted by FACS AriaII and washed with PBS.
Total RNA was isolated using RNeasy Mini kit (Qiagen). RNA was treated with TURBO DNase (Life Technologies). mRNA was purified from total RNA using Dynabeads mRNA purification kit (Life Technologies). cDNA was generated with the First-strand synthesis kit (Life Technologies) using a random hexamers in presence of actinomycin D. Second-strand synthesis was performed with dUTP instead of dTTP. Sonicated cDNA was ligated to adaptors (NEBNext primer kit). The resulting DNA was treated with uracil-N-glycolsylase prior to PCR amplification with the indexing primers (NEBNext primer kit).


title: MOLM13 METTL3 shRNA #12 Ribo-seq rep1

description:
ribosome-depleted RNA
rpf.counts.xls
m3kd.12.1.rpf


title: MOLM13 METTL3 shRNA #9 RNAseq rep 2

description:
ribosome-depleted RNA
rnaseq.counts.xls
ctrl2


title: AN20


title: RNAseq DN2 ThymoDp(A)/p(A) 1

description: used for Fig. 3


title: RNAseq DN2 WT 3

description: used for Fig. 3


title: RNAseq DN2 WT 2

description: used for Fig. 3


title: MOLM13 METTL3 shRNA #9 RNAseq rep 1

description:
ribosome-depleted RNA
rnaseq.counts.xls
ctrl1


title: MOLM13 METTL3 shRNA #12 RNAseq rep 3

description:
ribosome-depleted RNA
rnaseq.counts.xls
m3kd123


title: MOLM13 miCLIP rep 3

description: poly(A) RNA

source_name: MOLM13 poly(A) RNA

value: Rabbit polyclonal anti-N6-methyladenosine (m6A) (Abcam #ab151230)

value: NNNTGGCNN


title: RNAseq DN2 WT 1

description: used for Fig. 3


title: MOLM13 miCLIP rep 2

description: poly(A) RNA

value: NNNGTAANN


title: RN2_repA

data_processing:
Map to mm10 using STAR aligner
Duplicate removal using PICARD
Calculate counts for coding genes or catalog using htseq-count. To calculate the number of fragments mapping to each catalog transcript in each library, overlapping catalog genes were merged together
DESeq2 was used to calculate variance stabalized transformed values
Genome_build: mm10
Supplementary_files_format_and_content: all_hematopoiesis_catalog-vsd.csv: relative abundance estimations,mjdelasv_GEO_lowinputlibraries-merged_lncRNAassembly.gtf:lncRNA catalog assembly

source_name: AML cell line

extract_protocol:
Cells were sorted following previously published gating strategies, shown in Figure S1 and RNA was extracted using NucleoSpin RNA XS (Machery Nagel), including DNase treatment.
Between 3000 and 10,000 cells were used as library input using Clontech’s SMARTer Ultra Low input RNA Sequencing Kit (Cat. No. 634823). cDNA was sheared to 300 bp on a Covaris LE220 and used as input for Clontech’s low input library preparation for Illumina Sequencing kit (Cat. no. 634947). Libraries were sequenced paired-end 100 bp on an Illumina HiSeq2500.

tag: culture conditions

value: MLL-AF9;NRASG12D AML cells were obtained from the Lowe laboratory and cultured in RPMI-1640 with GlutaMax (Gibco), supplemented with 10% heat-inactivated FBS (HyClone) and 1% Penicillin/Streptomycin (Gibco) under 7.5% CO2 culture conditions.

value: Sorted cell culture cell line


title: AML-2_repB

source_name: Transplated AML sorted from bone marrow

value: Before transplantation, MLL-AF9;NRASG12D AML cells were obtained from the Lowe laboratory and cultured in RPMI-1640 with GlutaMax (Gibco), supplemented with 10% heat-inactivated FBS (HyClone) and 1% Penicillin/Streptomycin (Gibco) under 7.5% CO2 culture conditions.

value: Sorted AML cell from bone marrow 14 days post transplantation


title: MOLM13 miCLIP rep 1

description: poly(A) RNA

value: NNNTTAGNN


title: RN2_repB


title: AML-2_repA


title: ProB_repB

source_name: Primary bone marrow cells

tag: age

value: 8-12 weeks old

value: Sorted primary cells


title: PMA/Iono R3 ATAC-seq

city: Coventry

first: Nigel

middle: Paul

last: Dyer

address: Gibbet Hill Road

institute: University of Warwick

description: 20 ng/ml PMA and 1 ug/ml Iono for 3 hours

data_processing:
Bowtie2 using to align trimmed paired-end data and single-ended read data to the hs37d5 reference genome.
ATAC-seq peaks were identified using cisGenome, and peak sizes were claculated by determining the number of fragment ends that were located within each peak for each of the samples. Data was normlised by the linrary size for each sample.
Peak detection and coverage track generation were performed using MACS on merged replicates. Coverage tracks were uploaded to the UCSC Genome Browser.
Tag counts were normalized using centre-scaling, and sorted on log2 treatment/control fold change.
genome build: hs37d5
processed data files format and content: Data include all counts per peak per sample. A separate spreedsheet includes normalised peak counts per condition (of pooled replicates) used in the analyses.

source_name: Jurkat T cells

growth_protocol: Jurkat A3 T cells were routinely sub-cultured by plating 5 x 10^5 cells into a T75 flask every other day. Cells were cultured in RPMI media supplemented with 1% L-glutamine and 10% foetal calf serum. Cells were maintained at 37 degrees celcius and 5% CO2.

extract_protocol:
Nuclei were extracted from 50,000 cells using Nuclei EZ Lysis Buffer (Sigma-Aldrich).
ATAC libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Cells were treated with either 20 ng/ml PMA, 1 ug/ml Ionomycin, or 20 ng/ml PMA and 1 ug/ml Ionomycin for 3 hours.

tag: jurkat clone


title: PMA/Iono R2 ATAC-seq


title: PMA/Iono R1 ATAC-seq


title: RNAseq DN2 ThymoDp(A)/p(A) 3

description: used for Fig. 3


title: Iono R3 ATAC-seq

description: 1 ug/ml Iono for 3 hrs


title: ProB_repA


title: MOLM13 Control shRNA RNAseq rep 1

description:
ribosome-depleted RNA
rnaseq.counts.xls
m3kd91

value: Acute myeloid leukemia


title: Iono R2 ATAC-seq

description: 1 ug/ml Iono for 3 hrs


title: Iono R1 ATAC-seq

description: 1 ug/ml Iono for 3 hrs


title: PreB_repB


title: PreB_repA


title: PMA R2 ATAC-seq

description: 20 ng/ml PMA for 3 hrs


title: MEP_repB


title: PMA R3 ATAC-seq

description: 20 ng/ml PMA for 3 hrs


title: Gr1_repB


title: MEP_repA


title: Gr1_repA


title: Untreated R3 ATAC-seq

description: Untreated cells


title: Untreated R2 ATAC-seq

description: Untreated cells


title: GMP_repB


title: PMA R1 ATAC-seq

description: 20 ng/ml PMA for 3 hrs


title: GMP_repA


title: Untreated R1 ATAC-seq

description: Untreated cells


title: PMA/Iono R3 RNA-seq

data_processing:
Tophat (release 2.1.0) was used to align RNA-seq reads to the human reference genome (hs37d5).
mRNA expression levels were calculated per gene using htseq-count against release 19 of the comprehensive genome annotation from gencode.
Counts were normalized to adjust for the difference in total number of counts associated with each sample (library size) and pooled across triplicates.
Analysis focussed on genes that were differentially expressed by 2 or 5 fold in comparison to the unstimulated control.
genome build: hs37d5
processed data files format and content: Data include counts per gene per sample A separate spreadsheet includes  counts per contition (with pooled replicates) used in the analysis.

extract_protocol:
5 x 10^6 cells were lysed and RNA was extracted using a High Pure RNA Isolation kit (Roche).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cells were treated with either 30 ng/ml TNF, 20 ng/ml PMA, 1 ug/ml Ionomycin, or 20 ng/ml PMA and 1 ug/ml Ionomycin for 3 hours.


title: CMP_repB


title: PMA/Iono R1 RNA-seq


title: PMA/Iono R2 RNA-seq


title: Iono R3 RNA-seq

description: 1 ug/ml Iono for 3 hrs


title: CD3_repB


title: Iono R1 RNA-seq

description: 1 ug/ml Iono for 3 hrs


title: Iono R2 RNA-seq

description: 1 ug/ml Iono for 3 hrs


title: CLP_repA


title: LT-HSC_repB


title: CD3_repA


title: LT-HSC_repA


title: PMA R3 RNA-seq

description: 20 ng/ml PMA for 3 hrs


title: barc RNAi larval type I neuroblasts - replicate 1

city: Vienna

first: Thomas

middle: Rainer

last: Burkard

address: Dr. Bohrgasse 7

country: Austria

institute: IMBA

description: Sample NB 3 (internal ID 13190)

data_processing:
Basecalling RTA 1.18.64.0
The strand specific paired-end reads were screened for ribosomal RNA by aligning with BWA (v0.6.1) against known rRNA sequences (RefSeq). Non matching reads were considerd downstream.
The rRNA subtracted paired-end reads were aligned with TopHat (v1.4.1) against the Drosophila melanogaster genome (FlyBase r5.44) and a maxiumum of 6 missmatches.  Introns between 20-150000 bp are allowed which is based on FlyBase statistics. Maximum multihits was set to 1 and InDels as well as Microexon-search was enabled. Additionally, a gene model was provided as GTF (FlyBase r5.44).
Aligned reads in valid pairs are subjected to FPKM estimation with Cufflinks (v1.3.0). In this step bias detection and correction was performed. Furthermore, only those fragments compatible with FlyBase annotation (r5.44) were allowed and counted towards the number of mapped hits used in the FPKM denominator.  snRNA, rRNA, tRNA, snoRNA and pseudogenes are masked.
The aligned reads were counted with HTSeq. Counts for technical replicates are summed up. The polyA containing transcripts were subjected to differential expression analysis with DESeq. DESeq parameters were chosen to match the DESeq publication (Anders S, Huber W., 2010).
Genome_build: FlyBase dmel_r5.44
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample (Standard output cufflinks); tab-delimited text file with differentially expressed genes for the whole analysis (standard output DESeq).

molecule: polyA RNA

organism: Drosophila melanogaster

source_name: barc RNAi larval type I neuroblasts

growth_protocol: Flies expressing dicer2; asense-Gal4, UAS-CD8::GFP were crossed to flies with empty landing site VIE-260B (VDRC TID:60100) or barc RNAi (VDRC TID:107013) and the progeny was raised at 29C. Late 3rd instar larval brains were dissected and prepared for FACS-sorting. S2 cells were cultured in Schneider's medium + 10% heat-inactivated FBS and treated with dsRNA against mCherry or barc for 7days.

extract_protocol: Type I neuroblasts were FACS sorted from wild type (TID:60100) or barc RNAi, and RNA was isolated using Trizol LS reagent. S2 cells treated with dsRNA against mCherry or barc were washed and RNA was isolated using Trizol. For both experiments: Total RNA was enriched for polyA mRNA, fragmented and first strand and second strand (dUTP, instead dTTP was used) was generated. For the neuroblast sample, library preparation was done manually using the Illumina Kapa library prep kit, and for the S2 cell sample, library preparation was done with a robot using the Illumina NEB ultra Kit. For both samples, UGDase treatment was done to confer strand specificity.

tag: tissue

value: larval brain

value: Neuroblasts

tag: Stage

value: late third instar larva

tag: marker

value: cells express membrane-bound GFP marker (UAS-CD8::GFP)


title: wild type larval type I neuroblasts - replicate 2

description: Sample NB 2 (internal ID 13189)

source_name: wild type larval type I neuroblasts


title: wild type larval type I neuroblasts - replicate 1

description: Sample NB 1 (internal ID 13188)


title: barc RNAi larval type I neuroblasts - replicate 2

description: Sample NB 4 (internal ID 13191)


title: PMA R2 RNA-seq

description: 20 ng/ml PMA for 3 hrs


title: TNF R3 RNA-seq

description: 30 ng/ml TNF for 3 hrs


title: TNF R2 RNA-seq

description: 30 ng/ml TNF for 3 hrs


title: PMA R1 RNA-seq

description: 20 ng/ml PMA for 3 hrs


title: Untreated R3 RNA-seq

description: Untreated cells


title: Untreated R1 RNA-seq

description: Untreated cells


title: Untreated R2 RNA-seq

description: Untreated cells


title: TNF R1 RNA-seq

description: 30 ng/ml TNF for 3 hrs


title: CMP_repA


title: CLP_repB


title: PB-TERRA

city: Hefei

first: Xiaojuan

last: Xu

state: Anhui

address: 350 Shushanhu Road

institute: Hefei Institutes of Physical Science

description:
Inner cell mass of pre-implantation embryo.
Processed data file: PB-VS-PB-TERRA.PossionDis_Method.GeneDiffExp.txt

data_processing:
BGISEQ500 platform used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm9 whole genome using HISAT software.
Exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by Cufflinks.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: PB-VS-PB-TERRA.PossionDis_Method.GeneDiffExp.txt: Tab-delimited text file includes the expression values of this transcript for each Sample.

source_name: embryonic stem cells, PB-TERRA

growth_protocol: ESCs were routinely maintained on gelatin-coated plates in the conventional mouse ESC medium.

extract_protocol:
RNA was harvested using Trizol reagent. TruSeq® RNA LT Sample Prep Kit v2 was used with total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard BGISEQ500 protocols.

treatment_protocol: 46C mouse embryonic stem cells overexpressing PB-TERRA or PB empty vector.

tag: cell line

value: embryonic stem cell line

tag: overexpression

value: PB-TERRA

tag: passages


title: PB

source_name: embryonic stem cells, PB

tag: cell type

value: PB vector


title: budding yeast, in vivo, α-factor(−), 3% DMS

city: Fukuoka

first: Taichi

last: Umeyama

state: Fukuoka

address: Maidashi 3-1-1, Higashi-ku

department: School of Medicine

country: Japan

institute: Kyushu University

data_processing:
Library strategy: DMS-seq
Basecalls performed using CASAVA version 1.8.4
Sequenced reads were trimmed for adaptor sequence and masked for low-quality sequence, then mapped to S288C_reference_genome_R64-1-1_20110203 whole genome using bowtie v2.2.5 with parameters --no-mixed --no-discordant --very-sensitive
Genome_build: S288C_reference_genome_R64-1-1_20110203
Supplementary_files_format_and_content: bigWig

source_name: budding yeast

growth_protocol: Saccharomyces cerevisiae cells were cultivated in YPD medium (1% (w/v) yeast extract, 2% (w/v) polypepton, 2% (w/v) dextrose) at 30°C overnight, diluted 10-fold in fresh YPD medium, and further grown at 30°C for 3 h.

extract_protocol:
The cells were treated with Zymolyase, then incubated with ProK/SDS. The solution was extracted with phenol/chloroform and combined with ethanol and precipitated by centrifugation. The pelette was treated with RNaseA and the solution was extracted once with phenol/chloroform and once with chloroform. The solution was combined with ethanol  and precipitated by centrifugation, then dissolved in water.
DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo minus) and dATP to yield a protruding 3’- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 4–8 cycles.

treatment_protocol: The cells were collected by centrifugation and resuspended in the conditioned medium at ~1×109 cells/ml, then 1-ml aliquot of the yeast cell suspension was combined with 30 μl of DMS and incubated at room temperature for 2 min.

tag: strain

tag: in vivo/in vitro

value: in vivo


title: budding yeast, in vivo, α-factor(+), 3% DMS


title: PBS_Rep2_P3.S99

status: Public on Sep 17 2017

city: Cambridge

first: Samantha

last: Riesenfeld

state: MA

address: 415 Main St

institute: Broad Institute

data_processing:
Sequencing output of droplet-based 10X scRNA-seq samples was run through cellranger mkfastq, version 1.3.1, and the resulting fastq files were grouped by sample (across sequencing runs) and run through cellranger count, version 1.3.1, to obtain bam alignment files and gene count (UMI) matrices. The reference used is a version of mm10, modified so the 3' end of the Nmur1 gene extends to coordinate 86384000.
For plate-based scRNA-seq for WT and IL33, Rep1, genes were quantified using RSEM, version 1.2.8, by running rsem-calculate-expression with the option --bowtie-chunkmbs 512 on the paired fastq files with mm10 as the reference.
For all other plate-based scRNA-seq, genes were quantified by running kallisto, version 0.42.3, on the paired fastq files, followed by tximport from the R package tximport version 1.0.3, both with reference mm10.
Genome_build: mm10
Supplementary_files_format_and_content: For droplet data, UMI count expression values for each gene (rows) in each cell (columns). For plate data, RSEM read counts and normalized TPM expression for each gene (rows) in each cell (columns).

source_name: Innate lymphoid cell

growth_protocol: not applicable

extract_protocol:
After lung dissociation using the GentleMACS lung dissociation kit (Miltenyi Biotec), single cell suspensions were incubated with CD90.2 MicroBeads (Miltenyi Biotec) on ice and enriched for CD90.2+ cells by magnetic separation using LS columns according to the manufacturer’s protocol. CD90.2+ lung cells were then stained on ice with antibodies for sorting. Innate lymphoid cells were defined as 7AAD- CD45+ CD90.2+ CD127+ Lineage (CD11b, CD11c, CD19, NK1.1, CD3ε, CD4, CD8a, TCRβ, TCRγδ)- cells and sorted on a BD FACS Aria (BD Biosciences).
For droplet-based scRNA-seq, sorted innate lymphoid cells were encapsulated into droplets, and libraries were prepared using the ChromiumTM Single Cell 3’ Reagent Kits v2 according to manufacturer’s protocol (10x Genomics). The generated scRNA-seq libraries were sequenced using a 75 cycle Nextseq 500 high output V2 kit. For full-length scRNA-seq, single ILCs were sorted into 96-well plates containing 5ul TCL Buffer (QIAGEN) with 1% 2-Mercaptoethanol, centrifuged and frozen at -80°C. SMART-Seq2 protocol was carried out as previously described by Picelli et al. with minor modifications in the reverse transcription step. cDNA was amplified with 22 cycles and tagmented with one-eighth of the standard Illumina NexteraXT reaction volume. Single-cell libraries were pooled and paired-end sequenced (38bp x 2) with a 75 cycle Nextseq 500 high output V2 kit.

treatment_protocol:
Intranasal administration of PBS daily for 3 consecutive days.
see treatment protocol for each sample above

value: Innate lymphoid cell

value: WT

tag: organ

value: Lung

tag: treatment

value: PBS

tag: scrna-seq method

value: plate-based (SMART-Seq2)

tag: replicate

tag: plate


title: PBS_Rep2_P3.S97

tag: genotype


title: PBS_Rep2_P3.S190


title: PBS_Rep2_P3.S191


title: PBS_Rep2_P3.S192


title: PBS_Rep2_P3.S188


title: PBS_Rep2_P3.S186


title: PBS_Rep2_P3.S187


title: PBS_Rep2_P3.S184


title: PBS_Rep2_P3.S183


title: PBS_Rep2_P3.S185


title: PBS_Rep2_P3.S179


title: PBS_Rep2_P3.S180


title: PBS_Rep2_P3.S176


title: PBS_Rep2_P3.S177


title: PBS_Rep2_P3.S178


title: PBS_Rep2_P3.S174


title: PBS_Rep2_P3.S172


title: PBS_Rep2_P3.S171


title: PBS_Rep2_P3.S175


title: PBS_Rep2_P3.S173


title: PBS_Rep2_P3.S167


title: PBS_Rep2_P3.S170


title: PBS_Rep2_P3.S169


title: PBS_Rep2_P3.S168


title: PBS_Rep2_P3.S189


title: PBS_Rep2_P3.S164


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title: PBS_Rep2_P3.S163


title: PBS_Rep2_P3.S162


title: PBS_Rep2_P3.S161


title: PBS_Rep2_P3.S159


title: PBS_Rep2_P3.S155


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title: PBS_Rep2_P3.S150


title: PBS_Rep2_P3.S147


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title: PBS_Rep2_P3.S144


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title: PBS_Rep2_P3.S98


title: PBS_Rep2_P3.S131


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title: PBS_Rep2_P3.S143


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title: PBS_Rep2_P3.S118


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title: PBS_Rep2_P3.S114


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title: PBS_Rep2_P3.S127


title: PBS_Rep2_P3.S110


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title: PBS_Rep2_P3.S160


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title: PBS_Rep1_P2.S288


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title: PBS_Rep1_P2.S211


title: PBS_Rep1_P2.S208


title: PBS_Rep1_P2.S206


title: PBS_Rep1_P2.S207


title: PBS_Rep1_P2.S209


title: PBS_Rep1_P2.S212


title: PBS_Rep1_P2.S210


title: PBS_Rep1_P2.S205


title: PBS_Rep1_P2.S204


title: PBS_Rep1_P2.S203


title: PBS_Rep1_P2.S202


title: PBS_Rep1_P2.S201


title: PBS_Rep1_P2.S224


title: PBS_Rep1_P2.S199


title: PBS_Rep1_P2.S200


title: PBS_Rep1_P2.S198


title: PBS_Rep1_P2.S197


title: PBS_Rep1_P2.S195


title: PBS_Rep1_P1.S95


title: PBS_Rep1_P1.S94


title: PBS_Rep1_P1.S93


title: PBS_Rep1_P2.S214


title: PBS_Rep1_P1.S91


title: PBS_Rep1_P2.S270


title: PBS_Rep1_P1.S96


title: PBS_Rep1_P1.S92


title: PBS_Rep1_P1.S89


title: PBS_Rep1_P1.S85


title: PBS_Rep1_P1.S88


title: PBS_Rep1_P1.S83


title: PBS_Rep1_P1.S84


title: PBS_Rep1_P1.S86


title: PBS_Rep1_P1.S81


title: PBS_Rep1_P1.S80


title: PBS_Rep1_P1.S78


title: PBS_Rep1_P1.S74


title: PBS_Rep1_P1.S77


title: PBS_Rep1_P1.S75


title: PBS_Rep1_P1.S79


title: PBS_Rep1_P1.S76


title: PBS_Rep1_P1.S73


title: PBS_Rep1_P2.S196


title: PBS_Rep1_P1.S72


title: PBS_Rep1_P1.S71


title: PBS_Rep1_P1.S67


title: PBS_Rep1_P1.S69


title: PBS_Rep1_P1.S68


title: PBS_Rep1_P1.S64


title: PBS_Rep1_P1.S87


title: PBS_Rep1_P1.S70


title: PBS_Rep1_P1.S63


title: PBS_Rep1_P1.S90


title: PBS_Rep1_P1.S65


title: PBS_Rep1_P1.S66


title: PBS_Rep1_P1.S82


title: PBS_Rep1_P1.S58


title: PBS_Rep1_P1.S60


title: PBS_Rep1_P1.S62


title: PBS_Rep1_P1.S59


title: PBS_Rep1_P1.S57


title: PBS_Rep1_P1.S55


title: PBS_Rep1_P1.S56


title: PBS_Rep1_P1.S52


title: PBS_Rep1_P1.S49


title: PBS_Rep1_P1.S51


title: PBS_Rep1_P1.S53


title: PBS_Rep1_P1.S50


title: PBS_Rep1_P1.S54


title: PBS_Rep1_P1.S47


title: PBS_Rep1_P1.S48


title: PBS_Rep1_P1.S46


title: PBS_Rep1_P1.S44


title: PBS_Rep1_P1.S42


title: PBS_Rep1_P1.S43


title: PBS_Rep1_P1.S45


title: PBS_Rep1_P1.S61


title: PBS_Rep1_P1.S40


title: PBS_Rep1_P1.S38


title: PBS_Rep1_P1.S33


title: PBS_Rep1_P1.S32


title: PBS_Rep1_P1.S34


title: PBS_Rep1_P1.S35


title: PBS_Rep1_P1.S28


title: PBS_Rep1_P1.S36


title: PBS_Rep1_P1.S27


title: PBS_Rep1_P1.S30


title: PBS_Rep1_P1.S37


title: PBS_Rep1_P1.S31


title: PBS_Rep1_P1.S29


title: PBS_Rep1_P1.S24


title: PBS_Rep1_P1.S25


title: PBS_Rep1_P1.S21


title: PBS_Rep1_P1.S23


title: PBS_Rep1_P1.S20


title: PBS_Rep1_P1.S22


title: PBS_Rep1_P1.S19


title: PBS_Rep1_P1.S41


title: PBS_Rep1_P1.S18


title: PBS_Rep1_P1.S17


title: PBS_Rep1_P1.S15


title: PBS_Rep1_P1.S39


title: PBS_Rep1_P1.S16


title: PBS_Rep1_P1.S14


title: PBS_Rep1_P1.S11


title: PBS_Rep1_P1.S12


title: PBS_Rep1_P1.S13


title: PBS_Rep1_P1.S9


title: PBS_Rep1_P1.S10


title: PBS_Rep1_P1.S8


title: PBS_Rep1_P1.S7


title: PBS_Rep1_P1.S26


title: PBS_Rep1_P1.S4


title: PBS_Rep1_P1.S3


title: IL33_Rep2_P1.S384

treatment_protocol:
Intranasal administration of IL-33 daily for 3 consecutive days.
see treatment protocol for each sample above


title: PBS_Rep1_P1.S6


title: PBS_Rep1_P1.S5


title: IL33_Rep2_P1.S380


title: IL33_Rep2_P1.S382


title: IL33_Rep2_P1.S383


title: IL33_Rep2_P1.S377


title: IL33_Rep2_P1.S376


title: IL33_Rep2_P1.S378


title: IL33_Rep2_P1.S375


title: IL33_Rep2_P1.S371


title: IL33_Rep2_P1.S370


title: IL33_Rep2_P1.S372


title: IL33_Rep2_P1.S368


title: IL33_Rep2_P1.S367


title: IL33_Rep2_P1.S369


title: IL33_Rep2_P1.S366


title: IL33_Rep2_P1.S365


title: IL33_Rep2_P1.S364


title: IL33_Rep2_P1.S363


title: IL33_Rep2_P1.S362


title: IL33_Rep2_P1.S361


title: IL33_Rep2_P1.S360


title: IL33_Rep2_P1.S358


title: IL33_Rep2_P1.S379


title: IL33_Rep2_P1.S352


title: IL33_Rep2_P1.S351


title: IL33_Rep2_P1.S350


title: IL33_Rep2_P1.S353


title: IL33_Rep2_P1.S354


title: IL33_Rep2_P1.S355


title: IL33_Rep2_P1.S357


title: IL33_Rep2_P1.S356


title: IL33_Rep2_P1.S349


title: IL33_Rep2_P1.S346


title: IL33_Rep2_P1.S347


title: IL33_Rep2_P1.S381


title: IL33_Rep2_P1.S345


title: IL33_Rep2_P1.S344


title: IL33_Rep2_P1.S343


title: IL33_Rep2_P1.S341


title: IL33_Rep2_P1.S342


title: IL33_Rep2_P1.S337


title: IL33_Rep2_P1.S338


title: IL33_Rep2_P1.S359


title: IL33_Rep2_P1.S335


title: IL33_Rep2_P1.S340


title: IL33_Rep2_P1.S339


title: IL33_Rep2_P1.S334


title: IL33_Rep2_P1.S336


title: IL33_Rep2_P1.S333


title: IL33_Rep2_P1.S331


title: IL33_Rep2_P1.S348


title: IL33_Rep2_P1.S332


title: IL33_Rep2_P1.S324


title: IL33_Rep2_P1.S328


title: IL33_Rep2_P1.S320


title: IL33_Rep2_P1.S319


title: IL33_Rep2_P1.S325


title: IL33_Rep2_P1.S326


title: IL33_Rep2_P1.S322


title: IL33_Rep2_P1.S317


title: IL33_Rep2_P1.S329


title: IL33_Rep2_P1.S318


title: IL33_Rep2_P1.S314


title: IL33_Rep2_P1.S315


title: IL33_Rep2_P1.S330


title: IL33_Rep2_P1.S323


title: IL33_Rep2_P1.S316


title: IL33_Rep2_P1.S313


title: IL33_Rep2_P1.S309


title: IL33_Rep2_P1.S311


title: IL33_Rep2_P1.S307


title: IL33_Rep2_P1.S306


title: IL33_Rep2_P1.S302


title: IL33_Rep2_P1.S327


title: IL33_Rep2_P1.S305


title: IL33_Rep2_P1.S321


title: IL33_Rep2_P1.S303


title: IL33_Rep2_P1.S304


title: IL33_Rep2_P1.S300


title: IL33_Rep2_P1.S299


title: IL33_Rep2_P1.S298


title: IL33_Rep2_P1.S310


title: IL33_Rep2_P1.S312


title: IL33_Rep2_P1.S308


title: IL33_Rep2_P1.S294


title: IL33_Rep2_P1.S295


title: IL33_Rep2_P1.S290


title: IL33_Rep2_P1.S291


title: IL33_Rep2_P1.S297


title: IL33_Rep2_P1.S293


title: IL33_Rep2_P1.S292


title: IL33_Rep1_P2.S192


title: IL33_Rep2_P1.S289


title: IL33_Rep1_P2.S190


title: IL33_Rep1_P2.S191


title: IL33_Rep1_P2.S186


title: IL33_Rep1_P2.S187


title: IL33_Rep1_P2.S188


title: IL33_Rep1_P2.S185


title: IL33_Rep1_P2.S184


title: IL33_Rep2_P1.S301


title: IL33_Rep1_P2.S182


title: IL33_Rep1_P2.S183


title: IL33_Rep2_P1.S296


title: IL33_Rep1_P2.S181


title: IL33_Rep1_P2.S180


title: IL33_Rep1_P2.S178


title: IL33_Rep1_P2.S179


title: IL33_Rep1_P2.S177


title: IL33_Rep1_P2.S176


title: IL33_Rep1_P2.S175


title: IL33_Rep1_P2.S189


title: IL33_Rep1_P2.S171


title: IL33_Rep1_P2.S173


title: IL33_Rep1_P2.S170


title: IL33_Rep1_P2.S174


title: IL33_Rep1_P2.S172


title: IL33_Rep1_P2.S167


title: IL33_Rep1_P2.S169


title: IL33_Rep1_P2.S164


title: IL33_Rep1_P2.S163


title: IL33_Rep1_P2.S165


title: IL33_Rep1_P2.S166


title: IL33_Rep1_P2.S161


title: IL33_Rep1_P2.S162


title: IL33_Rep1_P2.S160


title: IL33_Rep1_P2.S159


title: IL33_Rep1_P2.S158


title: IL33_Rep1_P2.S156


title: IL33_Rep1_P2.S155


title: IL33_Rep1_P2.S157


title: IL33_Rep1_P2.S154


title: IL33_Rep1_P2.S153


title: IL33_Rep1_P2.S152


title: IL33_Rep1_P2.S151


title: IL33_Rep1_P2.S150


title: IL33_Rep1_P2.S146


title: IL33_Rep1_P2.S148


title: IL33_Rep1_P2.S168


title: IL33_Rep1_P2.S149


title: IL33_Rep1_P2.S147


title: IL33_Rep1_P2.S142


title: IL33_Rep1_P2.S144


title: IL33_Rep1_P2.S139


title: IL33_Rep1_P2.S138


title: IL33_Rep1_P2.S143


title: IL33_Rep1_P2.S141


title: IL33_Rep1_P2.S140


title: IL33_Rep1_P2.S133


title: IL33_Rep1_P2.S137


title: IL33_Rep1_P2.S136


title: IL33_Rep1_P2.S132


title: IL33_Rep1_P2.S130


title: IL33_Rep1_P2.S131


title: IL33_Rep1_P2.S126


title: IL33_Rep1_P2.S125


title: IL33_Rep1_P2.S127


title: IL33_Rep1_P2.S129


title: IL33_Rep1_P2.S128


title: IL33_Rep1_P2.S124


title: IL33_Rep1_P2.S145


title: IL33_Rep1_P2.S115


title: IL33_Rep1_P2.S116


title: IL33_Rep1_P2.S120


title: IL33_Rep1_P2.S121


title: IL33_Rep1_P2.S119


title: IL33_Rep1_P2.S117


title: IL33_Rep1_P2.S118


title: IL33_Rep1_P2.S112


title: IL33_Rep1_P2.S113


title: IL33_Rep1_P2.S114


title: IL33_Rep1_P2.S134


title: IL33_Rep1_P2.S135


title: IL33_Rep1_P2.S111


title: IL33_Rep1_P2.S109


title: IL33_Rep1_P2.S107


title: IL33_Rep1_P2.S108


title: IL33_Rep1_P2.S105


title: IL33_Rep1_P2.S106


title: IL33_Rep1_P2.S104


title: IL33_Rep1_P2.S122


title: IL33_Rep1_P2.S101


title: IL33_Rep1_P2.S102


title: IL33_Rep1_P2.S100


title: IL33_Rep1_P2.S123


title: IL33_Rep1_P2.S103


title: IL33_Rep1_P2.S99


title: IL33_Rep1_P1.S384


title: IL33_Rep1_P1.S383


title: IL33_Rep1_P1.S380


title: IL33_Rep1_P1.S381


title: IL33_Rep1_P1.S377


title: IL33_Rep1_P1.S379


title: IL33_Rep1_P1.S378


title: IL33_Rep1_P1.S373


title: IL33_Rep1_P1.S375


title: IL33_Rep1_P1.S374


title: IL33_Rep1_P1.S372


title: IL33_Rep1_P1.S371


title: IL33_Rep1_P2.S110


title: IL33_Rep1_P1.S370


title: IL33_Rep1_P1.S369


title: IL33_Rep1_P1.S366


title: IL33_Rep1_P1.S367


title: IL33_Rep1_P1.S365


title: IL33_Rep1_P1.S364


title: IL33_Rep1_P1.S362


title: IL33_Rep1_P1.S359


title: IL33_Rep1_P1.S360


title: IL33_Rep1_P1.S363


title: IL33_Rep1_P1.S361


title: IL33_Rep1_P1.S376


title: IL33_Rep1_P1.S382


title: IL33_Rep1_P1.S355


title: IL33_Rep1_P1.S358


title: IL33_Rep1_P1.S356


title: IL33_Rep1_P1.S357


title: IL33_Rep1_P1.S352


title: IL33_Rep1_P1.S368


title: IL33_Rep1_P1.S354


title: IL33_Rep1_P1.S353


title: IL33_Rep1_P1.S349


title: IL33_Rep1_P1.S348


title: IL33_Rep1_P1.S350


title: IL33_Rep1_P1.S351


title: IL33_Rep1_P1.S345


title: IL33_Rep1_P1.S344


title: IL33_Rep1_P1.S346


title: IL33_Rep1_P1.S339


title: IL33_Rep1_P1.S338


title: IL33_Rep1_P1.S342


title: IL33_Rep1_P1.S341


title: IL33_Rep1_P1.S340


title: IL33_Rep1_P1.S335


title: IL33_Rep1_P1.S334


title: IL33_Rep1_P1.S331


title: IL33_Rep1_P1.S332


title: IL33_Rep1_P1.S337


title: IL33_Rep1_P1.S330


title: IL33_Rep1_P1.S329


title: IL33_Rep1_P1.S333


title: IL33_Rep1_P1.S336


title: IL33_Rep1_P1.S347


title: IL33_Rep1_P1.S326


title: IL33_Rep1_P1.S343


title: IL33_Rep1_P1.S328


title: IL33_Rep1_P1.S325


title: IL33_Rep1_P1.S320


title: IL33_Rep1_P1.S323


title: IL33_Rep1_P1.S321


title: IL33_Rep1_P1.S324


title: IL33_Rep1_P1.S318


title: IL33_Rep1_P1.S319


title: IL33_Rep1_P1.S317


title: IL33_Rep1_P1.S316


title: IL33_Rep1_P1.S314


title: IL33_Rep1_P1.S315


title: IL33_Rep1_P1.S327


title: IL33_Rep1_P1.S310


title: IL33_Rep1_P1.S312


title: IL33_Rep1_P1.S308


title: IL33_Rep1_P1.S309


title: IL33_Rep1_P1.S311


title: IL33_Rep1_P1.S304


title: IL33_Rep1_P1.S306


title: IL33_Rep1_P1.S302


title: IL33_Rep1_P1.S303


title: IL33_Rep1_P1.S307


title: IL33_Rep1_P1.S322


title: IL33_Rep1_P1.S301


title: IL33_Rep1_P1.S305


title: IL33_Rep1_P1.S300


title: IL33_Rep1_P1.S299


title: IL33_Rep1_P1.S298


title: IL33_Rep1_P1.S297


title: IL33_Rep1_P1.S296


title: IL33_Rep1_P1.S295


title: IL33_Rep1_P1.S293


title: IL25_Rep2_P1.S288

treatment_protocol:
Intranasal administration of IL-25 daily for 3 consecutive days.
see treatment protocol for each sample above


title: IL33_Rep1_P1.S292


title: IL33_Rep1_P1.S291


title: IL25_Rep2_P1.S287


title: IL33_Rep1_P1.S313


title: IL25_Rep2_P1.S285


title: IL25_Rep2_P1.S284


title: IL25_Rep2_P1.S282


title: IL25_Rep2_P1.S281


title: IL25_Rep2_P1.S280


title: IL25_Rep2_P1.S276


title: IL25_Rep2_P1.S275


title: IL25_Rep2_P1.S274


title: IL33_Rep1_P1.S294


title: IL25_Rep2_P1.S269


title: IL25_Rep2_P1.S271


title: IL25_Rep2_P1.S273


title: IL25_Rep2_P1.S268


title: IL25_Rep2_P1.S272


title: IL25_Rep2_P1.S270


title: IL25_Rep2_P1.S265


title: IL25_Rep2_P1.S263


title: IL25_Rep2_P1.S262


title: IL25_Rep2_P1.S267


title: IL25_Rep2_P1.S266


title: IL25_Rep2_P1.S260


title: IL25_Rep2_P1.S264


title: IL25_Rep2_P1.S286


title: IL25_Rep2_P1.S258


title: IL25_Rep2_P1.S261


title: IL25_Rep2_P1.S259


title: IL25_Rep2_P1.S257


title: IL25_Rep2_P1.S256


title: IL25_Rep2_P1.S279


title: IL25_Rep2_P1.S255


title: IL25_Rep2_P1.S254


title: IL25_Rep2_P1.S253


title: IL25_Rep2_P1.S252


title: IL25_Rep2_P1.S249


title: IL25_Rep2_P1.S250


title: IL25_Rep2_P1.S251


title: IL25_Rep2_P1.S248


title: IL25_Rep2_P1.S247


title: IL25_Rep2_P1.S246


title: IL25_Rep2_P1.S245


title: IL25_Rep2_P1.S244


title: IL25_Rep2_P1.S241


title: IL25_Rep2_P1.S239


title: IL25_Rep2_P1.S240


title: IL25_Rep2_P1.S238


title: IL25_Rep2_P1.S236


title: IL25_Rep2_P1.S234


title: IL25_Rep2_P1.S243


title: IL25_Rep2_P1.S235


title: IL25_Rep2_P1.S231


title: IL25_Rep2_P1.S230


title: IL25_Rep2_P1.S242


title: IL25_Rep2_P1.S233


title: IL25_Rep2_P1.S227


title: IL25_Rep2_P1.S226


title: IL25_Rep2_P1.S232


title: IL25_Rep2_P1.S224


title: IL25_Rep2_P1.S225


title: IL25_Rep2_P1.S222


title: IL25_Rep2_P1.S221


title: IL25_Rep2_P1.S237


title: IL25_Rep2_P1.S223


title: IL25_Rep2_P1.S218


title: IL25_Rep2_P1.S219


title: IL25_Rep2_P1.S216


title: IL25_Rep2_P1.S214


title: IL25_Rep2_P1.S220


title: IL25_Rep2_P1.S215


title: IL25_Rep2_P1.S211


title: IL25_Rep2_P1.S213


title: IL25_Rep2_P1.S229


title: IL25_Rep2_P1.S209


title: IL25_Rep2_P1.S210


title: IL25_Rep2_P1.S208


title: IL25_Rep2_P1.S204


title: IL25_Rep2_P1.S207


title: IL25_Rep2_P1.S212


title: IL25_Rep2_P1.S206


title: IL25_Rep2_P1.S205


title: IL25_Rep2_P1.S203


title: IL25_Rep2_P1.S201


title: IL25_Rep2_P1.S200


title: IL25_Rep2_P1.S199


title: IL25_Rep2_P1.S217


title: IL25_Rep2_P1.S196


title: IL25_Rep2_P1.S197


title: IL25_Rep2_P1.S195


title: IL25_Rep2_P1.S198


title: IL25_Rep2_P1.S194


title: IL25_Rep2_P1.S193


title: IL25_Rep1_P2.S382


title: IL25_Rep1_P2.S381


title: IL25_Rep1_P2.S380


title: IL25_Rep1_P2.S377


title: IL25_Rep1_P2.S376


title: IL25_Rep2_P1.S202


title: IL25_Rep1_P2.S378


title: IL25_Rep1_P2.S375


title: IL25_Rep1_P2.S372


title: IL25_Rep1_P2.S368


title: IL25_Rep1_P2.S371


title: IL25_Rep1_P2.S369


title: IL25_Rep1_P2.S370


title: IL25_Rep1_P2.S367


title: IL25_Rep1_P2.S366


title: IL25_Rep1_P2.S365


title: IL25_Rep1_P2.S364


title: IL25_Rep1_P2.S363


title: IL25_Rep1_P2.S384


title: IL25_Rep1_P2.S383


title: IL25_Rep1_P2.S362


title: IL25_Rep1_P2.S358


title: IL25_Rep1_P2.S360


title: IL25_Rep1_P2.S379


title: IL25_Rep1_P2.S357


title: IL25_Rep1_P2.S356


title: IL25_Rep1_P2.S355


title: IL25_Rep1_P2.S354


title: IL25_Rep1_P2.S353


title: IL25_Rep1_P2.S352


title: IL25_Rep1_P2.S351


title: IL25_Rep1_P2.S350


title: IL25_Rep2_P1.S283


title: IL25_Rep1_P2.S361


title: IL25_Rep1_P2.S349


title: IL25_Rep1_P2.S359


title: IL25_Rep1_P2.S347


title: IL25_Rep1_P2.S348


title: IL25_Rep1_P2.S344


title: IL25_Rep1_P2.S345


title: IL25_Rep1_P2.S340


title: IL25_Rep1_P2.S342


title: IL25_Rep1_P2.S338


title: IL25_Rep1_P2.S339


title: IL25_Rep1_P2.S343


title: IL25_Rep1_P2.S341


title: IL25_Rep1_P2.S337


title: IL25_Rep2_P1.S228


title: IL25_Rep1_P2.S336


title: IL25_Rep1_P2.S334


title: IL25_Rep1_P2.S335


title: IL25_Rep1_P2.S333


title: IL25_Rep1_P2.S331


title: IL25_Rep1_P2.S330


title: IL25_Rep1_P2.S332


title: IL25_Rep1_P2.S346


title: IL25_Rep1_P2.S328


title: IL25_Rep1_P2.S326


title: IL25_Rep1_P2.S327


title: IL25_Rep1_P2.S329


title: IL25_Rep1_P2.S325


title: IL25_Rep1_P2.S324


title: IL25_Rep1_P2.S323


title: IL25_Rep1_P2.S317


title: IL25_Rep1_P2.S319


title: IL25_Rep1_P2.S314


title: IL25_Rep1_P2.S316


title: IL25_Rep1_P2.S318


title: IL25_Rep1_P2.S321


title: IL25_Rep1_P2.S320


title: IL25_Rep1_P2.S310


title: IL25_Rep1_P2.S312


title: IL25_Rep1_P2.S315


title: IL25_Rep1_P2.S313


title: IL25_Rep1_P2.S311


title: IL25_Rep1_P2.S309


title: IL25_Rep1_P2.S308


title: IL25_Rep1_P2.S307


title: IL25_Rep1_P2.S306


title: IL25_Rep1_P2.S305


title: IL25_Rep1_P2.S303


title: IL25_Rep1_P2.S304


title: IL25_Rep1_P2.S301


title: IL25_Rep1_P2.S300


title: IL25_Rep1_P2.S298


title: IL25_Rep1_P2.S322


title: SU-DHL-4_DMSO_168h_rep3

first: Daniel

last: Gerlach

address: Dr.-Boehringer-Gasse 5-11

department: Global Computational Biology and Digital Sciences

institute: Boehringer Ingelheim RCV GmbH & Co KG

data_processing:
Base calling was performed using Illumina RTA 1.18.64, afterwards multiplexed samples were converted to FASTQ files using CASAVA 1.8.2.
Reads were mapped to the human genome assembly hg19 (hs37d5) with gsnap version 2012-12-20. The ENSEMBL 70 gene annotation was used for further expression estimations. Raw counts were computed using HTSeq version 0.5.3p9, FPKM expression values were estimated using Cufflinks version 2.0.2.
Genome_build: hg19
Supplementary_files_format_and_content: Raw counts expression values in matrix format (tab characater separated)

growth_protocol: Cells were kept in RPMI (ATCC, #30-2001). DMSO or compound from 1 mM stocks were added to cells at a density of 1 million cells per ml. For 7 day (168h) treatments, cells were split once after 3 days to 1 million cells per ml, and fresh compound or DMSO were added.

extract_protocol:
Cells were lysed in TRI lysis reagent (Sigma, T9424).
Approximately 500ng of total RNA were subjected to library preparation using the TruSeq RNA Library Prep Kit v2 with polyA selection as recommended by the manufacturer (Illumina) without generating stranded information.

treatment_protocol: Compounds were added directly to the cells from 1 mM stock solutions in DMSO.

value: lymphoma cell line

tag: treated with


title: SU-DHL-4_DMSO_20h_rep2


title: SU-DHL-4_DMSO_20h_rep3


title: SU-DHL-4_DMSO_20h_rep1


title: IL25_Rep1_P2.S296


title: SU-DHL-4_DMSO_168h_rep1


title: IL25_Rep1_P2.S297


title: SU-DHL-4_BI00923812_20h_rep3


title: SU-DHL-4_BI00923812_20h_rep1


title: IL25_Rep1_P2.S293


title: IL25_Rep1_P2.S295


title: IL25_Rep1_P2.S294


title: SU-DHL-4_BI00923812_20h_rep2


title: IL25_Rep1_P2.S291


title: IL25_Rep1_P2.S292


title: SU-DHL-4_BI00923812_168h_rep1


title: SU-DHL-4_BI00923812_168h_rep2


title: IL25_Rep1_P2.S302


title: IL25_Rep1_P2.S289


title: HDM_Nmur1 KO_10x_rep2

treatment_protocol:
Intranasal administration of house dust mite (HDM) on day 0, 7, 8 and 9.
see treatment protocol for each sample above

value: Nmur1 KO

value: House dust mite (HDM)

value: Replicate 2


title: IL25_Rep1_P2.S299


title: SU-DHL-4_BI00923802_20h_rep2


title: SU-DHL-4_BI00923802_20h_rep3


title: HDM_Nmur1 KO_10x_rep1

value: Replicate 1


title: PBS ctrl for HDM_Nmur1 KO_10x_rep2

treatment_protocol:
Intranasal administration of PBS on day 0, 7, 8 and 9.
see treatment protocol for each sample above


title: SU-DHL-4_DMSO_168h_rep2


title: SU-DHL-4_BI00923802_168h_rep3


title: SU-DHL-4_BI00923802_168h_rep2


title: SU-DHL-4_BI00923802_168h_rep1


title: Farage_DMSO_20h_rep3

value: Farage


title: SU-DHL-4_BI00923812_168h_rep3


title: IL25_Rep1_P2.S290


title: PBS ctrl for HDM_WT_10x_rep2


title: PBS ctrl for HDM_Nmur1 KO_10x_rep1


title: HDM_WT_10x_rep2


title: Farage_DMSO_168h_rep3


title: PBS ctrl for HDM_WT_10x_rep1


title: NMU+IL-25_WT_10x_rep2

treatment_protocol:
Intranasal administration of NMU+IL-25 daily for 3 consecutive days.
see treatment protocol for each sample above


title: NMU_WT_10x_rep2

treatment_protocol:
Intranasal administration of NMU daily for 3 consecutive days.
see treatment protocol for each sample above

value: NMU


title: Farage_DMSO_20h_rep1


title: NMU+IL-25_WT_10x_rep1


title: Farage_BI00923812_20h_rep3


title: Farage_DMSO_168h_rep2


title: Farage_DMSO_168h_rep1


title: Farage_BI00923812_20h_rep2


title: SU-DHL-4_BI00923802_20h_rep1


title: IL-33_WT_10x_rep1


title: NMU_WT_10x_rep1


title: IL-33_WT_10x_rep2


title: IL-25_WT_10x_rep2


title: PBS_Rep1_P2.S265


title: IL-25_WT_10x_rep1


title: Farage_BI00923812_168h_rep3


title: Farage_BI00923812_168h_rep2


title: HDM_WT_10x_rep1


title: Farage_DMSO_20h_rep2


title: Farage_BI00923812_168h_rep1


title: PBS ctrl for alarmins_WT_10x_rep1


title: Farage_BI00923802_20h_rep3


title: Farage_BI00923802_168h_rep3


title: Farage_BI00923802_20h_rep2


title: Farage_BI00923802_20h_rep1


title: Farage_BI00923812_20h_rep1


title: Farage_BI00923802_168h_rep2


title: Farage_BI00923802_168h_rep1


title: Qkiac 3

city: PARIS

first: sophie

last: lemoine

address: 46 rue d'Ulm

department: Biologie

country: France

institute: Ecole Normale Supérieure

data_processing:
Before mapping, poly N read tails were trimmed, reads ≤40 bases were removed, and reads with quality mean ≤30 were discarded.
To obtain the counts on the exon features, reads were then aligned against the genome using Bowtie (version 0.12.9) with arguments -n 2 -l 34 -e 70 -k 2 --best.
To obtain the counts on the gene features, reads were then aligned against the genome using STAR (version 2.4.0j) and Ensembl annotation v78.
Alignments from reads matching more than once on the reference genome were removed using Java version of samtools.
To compute gene expression, Danio rerio Zv9 GFF3 genome annotation version 78 from Ensembl database was used.
All overlapping regions between alignments and referenced exons were counted using HTSeq-count 0.5.3.
To obtain the counts on the exon features, we used the bowtie alignments, excluding the junction reads. HTSeq-count was used with arguments: genomictype=exon, attributeid=Name, stranded=no, overlapmode=union, removeambiguouscases=false.
To obtain the counts on the gene features, we used the STAR alignments, including the junction reads. HTSeq-count was used with arguments: genomictype=exon, stranded=no, overlapmode=union, removeambiguouscases=false and attributeid=Alias where Alias was the corresponding parent gene ID.
The sample counts were normalized using DESeq 1.8.3.
Statistical treatments and differential analyses were also performed using DESeq 1.8.3 with arguments: disp.est.method=pooled, disp.est.sharing.mode=maximum, disp.est.fit.type=parametric
Genome_build: Danio rerio Zv9
Supplementary_files_format_and_content: tab-delimited text files include normalized reads counts for each Sample using DESeq 1.8.3 with arguments: disp.est.method=pooled, disp.est.sharing.mode=maximum, disp.est.fit.type=parametric.

organism: Danio rerio

source_name: trunk

growth_protocol:
Embryos resulting from crossing between qkia+/- fish were injected at one cell stage with qkic morpholino or control morpholino (0,6pmol).
We let them develop until 24-26hpf, stage where we could morphologically identify control embryos (sibling qkia+/+ or qkia+/- with control morpholino) and double qkia/c loss of function embryos (qkia-/- with qkic morpholino).

extract_protocol:
total RNA was extracted using the RNeasy Plus Universal Mini kit (Qiagen) and treated with Dnase.
Messenger (polyA+) RNAs were purified from 0.7 µg of total RNA using oligo(dT). Libraries were prepared using the strand non-specific RNA-Seq library preparation TruSeq RNA Sample Prep v2 kit (Illumina). Libraries were multiplexed by 6 on 2 flowcell lane(s). A 50 bp read sequencing was performed on a HiSeq 1500 device (Illumina). A mean of 27459238  ± 1611623 million passing Illumina quality filter reads was obtained for each of the 6 samples.

treatment_protocol: Zebrafish trunks at 24-26hpf were dissected (removal of head and yolk), flash-frozen on dry ice and stored at -80°C until RNA extraction.

value: embryo 24-26hpf

value: qkia-/-

tag: injection

value: qkic morpholino


title: Control 2

value: qkia+/+ or qkia+/-

value: control morpholino


title: Qkiac 2


title: Qkiac 1


title: Control 3


title: Importazole_rep3

status: Public on Sep 16 2017

city: Fujisawa

first: Yoshiyuki

last: Tsujihata

state: Kanagawa

address: 26-1, Muraoka-Higashi 2-chome

institute: Takeda Pharmaceutical Company Limited

data_processing:
Base calling was performed using software provided with the Ion Torrent Proton sequencer (Torrent suite ver.5.0.4).
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome reference using a combination of STAR (https://academic.oup.com/bioinformatics/article/29/1/15/272537/STAR-ultrafast-universal-RNA-seq-aligner), and Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml).
FPKM were calculated using the Cufflinks package (http://cufflinks.cbcb.umd.edu) and software provided with the Ion Torrent Proton sequencer (RNASeqAnalysis v5.0.3.0).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *txt: Tab-delimited text files include gene symbol and gene expression (FPKM) for each sample.

source_name: HL-1 cardiomyocytes

growth_protocol: HL-1 cardiomyocytes were grown in Claycomb Medium.

extract_protocol:
Total RNA was extracted using the RNeasy Mini kit (Qiagen).
mRNA were captured using Dynabeads® mRNA DIRECT™ Micro Purification Kit (Thermo Fisher Scientific, Waltham, MA) and used to generate sequencing libraries of barcoded fragment using the Ion Total RNA-Seq Core Kit v2 (Thermo Fisher Scientific) following the manufacturer's instructions. Libraries were sequenced on the Ion Proton™ System using the Ion PI™ Hi-Q Sequencing 200 Kit (Thermo Fisher Scientific).

treatment_protocol: The cells were treated with either vehicle (0.1% DMSO), 5 µM Ivermectin, or 10 µM Importazole for 24 hours.

value: cardiomyocyte cell line

tag: tissue origination

value: heart

value: 10 uM Importazole


title: Control 1

tag: Stage


title: Ivermectin_rep3

value: 5 uM Ivermectin


title: Ivermectin_rep1


title: Importazole_rep1


title: Ivermectin_rep2


title: Vehicle_rep3

value: 0.1% DMSO


title: Importazole_rep2


title: Vehicle_rep1


title: Vehicle_rep2


title: Endothelin-1 + Ivermectin_rep4

data_processing:
Base calling was performed using software provided with the Ion Torrent Proton sequencer (Torrent suite ver.5.0.4).
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19_AmpliSeq_Transcriptome_ERCC_v1 using software provided with the Ion Torrent Proton sequencer (AmpliSeqRNA v5.0.4.0).
RPM were calculated using software provided with the Ion Torrent Proton sequencer (AmpliSeqRNA v5.0.4.0).
Genome_build: hg19_AmpliSeq_Transcriptome_ERCC_v1
Supplementary_files_format_and_content: *txt: Tab-delimited text files include gene symbol and gene expression (RPM) for each sample.

source_name: iCell® Cardiomyocytes

growth_protocol: iCell® Cardiomyocytes (Cellular Dynamics International) were maintained in Maintenance Medium (Cellular Dynamics International).

extract_protocol:
Total RNA was extracted using QIAzol Lysis Reagent (Qiagen).
Whole transcriptome barcoded cDNA library is first generated with SuperScript® VILO™ cDNA Synthesis kit (Thermo Fisher Scientific) from 20 ng of total RNA. Then cDNA is amplified using Ion AmpliSeq™ technology to accurately maintain expression levels of all targeted genes. Amplified cDNA Libraries were evaluated for quality and quantified using Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). Libraries were then diluted to 75 pM and pooled equally, with fourteen individual samples per pool.

treatment_protocol: The cardiomyocytes were untreated or treated with endothelin-1 in the presence of either vehicle (DMSO), Ivermectin, Importazole, IPA-3, or verapamil for 18 hours.

value: derived cardiomyocytes from hiPSC

value: 10 nM Endothelin-1 + 3 uM Ivermectin


title: PBS ctrl for alarmins_WT_10x_rep2

value: Innate lymphoid cell

value: WT

value: Lung

value: PBS


title: Endothelin-1 + Ivermectin_rep3


title: Endothelin-1 + IPA-3_rep4

value: 10 nM Endothelin-1 + 10 uM IPA-3


title: Endothelin-1 + Ivermectin_rep1


title: Endothelin-1 + Ivermectin_rep2


title: Endothelin-1 + IPA-3_rep3


title: Endothelin-1 + IPA-3_rep2


title: Endothelin-1 + IPA-3_rep1


title: Endothelin-1 + Importazole_rep2

value: 10 nM Endothelin-1 + 10 uM Importazole


title: Endothelin-1 + Importazole_rep1


title: Endothelin-1 + Importazole_rep3


title: Endothelin-1 + Verapamil_rep2

value: 10 nM Endothelin-1 + 1 uM Verapamil


title: Endothelin-1 + Vehicle_rep1

value: 10 nM Endothelin-1 + Vehicle (0.1% DMSO)


title: Endothelin-1 + Vehicle_rep3


title: Endothelin-1 + Vehicle_rep4


title: Unstimulated_rep3

value: Unstimulated


title: Unstimulated_rep1


title: 6628_LexAO_HDA1_K9me3_21617

city: Eugene

first: Jordan

middle: David

last: Gessaman

state: OR

address: 355 Streisinger Hall

department: Biology, Institute of Molecular Biology

institute: University of Oregon

description: Tethered HDA-1 with LexAO binding site integrated at his-3.

data_processing:
All processing steps were done utilizing the Galaxy bioinformatics suite (usegalaxy.org; Afgan et al. 2016). The raw data (fastq) files were converted to fastqsanger format using FASTQ groomer.
The converted fastq files were mapped to the Ncrassa12 genome using Bowtie2, generating .bam files
The bam files were then converted into tdf files using IGVtools.  The Ncrassa12 genome was used as a reference genome and window size was set to 1 bp.
Genome_build: Ncrassa version 12 (fixed for inversion on LG VI) with LexAO sequence integrated (nc12_lexao.txt)
Supplementary_files_format_and_content: The tdf files were generated using the "count" function in igvtools (Integrative Genomics Viewer; Broad Institute, Robinson et al, 2011), using a window size of 1bp.  The tdf files are binary files that shows enrichment peaks for each ChIP sample and have been processed for faster display of the data in IGV.

organism: Neurospora crassa

source_name: Mycelia

growth_protocol: Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC

extract_protocol:
Mycelia were cross-linked for 10 min in 0.5% formaldehyde for histone modification ChIP.  Tissue was disrupted by sonication for thirty pulses (2x 15 pulses) before chromatin was sheared using a Bioruptor (Diagenode) for 15 min with a cycle of 30 sec on followed by 30 sec off, at high power.  DNA was purified using the Qiagen Minelute reaction cleanup kit (Qiagen, 28606) and eluted in 50 μl of water.
Approximately 10 ng of DNA was used to generate ChIP-Seq libraries.  Each library was prepared using the NEBNext Ultra DNA library prep kit (NEB #E7370) according to the manufacturer’s instructions.  “Invisible” fragments between 225-425 bp were excised and purified using the MinElute gel extraction kit (Qiagen, 28606).  Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 9 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min.

treatment_protocol: Mycelia were washed in PBS prior to crosslinking

tag: chip antibody

value: anti-H3K9me3 (Active Motif, catalog# 39161, lot# 13509002)

value: his-3+::LexAO; HDA-1-LexADBD::hph


title: Unstimulated_rep4


title: 6622_LexAO_HP1_dHDA1_K9me3_21617

description: Tethered HP1 with LexAO binding site integrated at his-3 in a delta hda-1 background.

value: his-3+::LexAO; HP1-LexADBD::hph; delta hda-1::hph


title: 6166_LexAO_HP1_K9me3_21617

description: Tethered HP1 with LexAO binding site integrated at his-3.

value: his-3+::LexAO; HP1-LexADBD::hph


title: Endothelin-1 + Verapamil_rep3


title: 6623_LexAO_HP1_dDIM2_K9me3_21617

description: Tethered HP1 with LexAO binding site integrated at his-3 in a delta dim-2 background.

value: his-3+::LexAO; HP1-LexADBD::hph; delta dim-2::hph


title: Nodule_dctA-_maeP_1

city: Hamilton

first: Turlough

middle: M

last: Finan

state: Ontario

address: 1280 Main St. W.

department: Department of Biology

country: Canada

institute: McMaster University

data_processing:
Quality control:  The absence of adapter sequences in the raw reads, the high quality at all nucleotide positions of the reads, and the absence of low quality reads were confirmed with FastQC.
Read mapping: Raw sequencing reads were mapped to a fasta file containing the reference sequences of both the plant and bacterial partners (the file is described in the genome build field) using Bowtie 2 with default settings, a read count summary table was prepared with HTSeq-count using the ‘-a 0’ option.
Differential expression: Differential expression was determined using DeSeq2 with default settings in R, considering the plant and bacterial features together.
Genome_build: The annotated P. sativum cv. Camphor transcriptome (Alves-Carvalho et al. 2015. Plant J. 84:1–19) was downloaded, and the high and low copy files were concatenated into a single fasta file.  The nucleotide fasta file of the coding regions of the Genbank assembly (GCA_000009265.1_ASM926v1) of the R. leguminosarum bv. viciae 3841 genome (Young et al. 2006. Genome Biol. 7:R34) was also downloaded.  All nucleotide sequences from both organisms were combined into a single contiguous nucleotide fasta file, and a gff file indicating the location of all plant and bacterial features was prepared.  In total, 53,171 features were considered: 45,908 P. sativum transcripts and 7,263 R. leguminosarum coding regions.
Supplementary_files_format_and_content: Combined_processed_data_plant.xlsx: Contains gene information and the DESeq2 output for each unigene in the P. sativum transcriptome.  Columns include: Gene (transcript unique identifier), MercatorBin (the functional annotation of the gene as determined with Mercator), eggnogAnnotation (the functional annotation of the gene as determined with eggnog-mapper), and several DESeq2 output values (baseMean, log2FoldChange, lfcSE, STAT, pvalue, padj)
Supplementary_files_format_and_content: Combined_processed_data_bacterial.xlsx: Contains gene information and the DESeq2 output for each protein coding gene in the R. leguminosarum bv. viciae genome.  Columns include: GeneID (the GeneID of the gene in the gff file), ProteinAccession (the protein accession number), LocusTag (the gene's unique locus tag), GeneName (the name of the gene), Function (the annotated function of the gene), and several DESeq2 output values (baseMean, log2FoldChange, lfcSE, STAT, pvalue, padj)

organism: Lathyrus oleraceus

source_name: Plant nodule tissue infected with bacterial cells.

growth_protocol: Sterile seeds were germinated on 1% water agar plates for 72 hours at room temperature in the dark.  Three seedlings were planted per Leonard assembly (Magenta jars containing a 1:1 [w/w] misture of quartz sand / vermiculite, and 250 mL of Jensen’s medium [per litre: 1 g CaHPO4, 0.2 g K2HPO4, 0.2 g MgSO4x7H2O, 0.2 g NaCl, 0.1 g FeCl2, 1 mg H3BO3, 1 mg ZnSO4x7H2O, 0.5 mg CuSO4x5H2O, 0.5 mg MnCl2x4H2O, 1 mg Na2MoO4x2H2O, 10 mg Na2EDTA, 2 mg NaFeEDTA, 0.4 mg biotin]) and inoculated with ~ 1 x 108 colony forming units of the appropriate R. leguminosarum strain 48 hours later.  Plants were grown in a Conviron growth chamber with a day (18 h, 218C) and night (6 h, 178C) cycle.  Plants were routinely watered with sterile distilled water, and once with 200 mL of sterile Jensen’s media 21 days post-inoculation with R. leguminosarum.  Nodules were collected 25 days post-inoculation with R. leguminosarum.

extract_protocol:
Nodules were collected from pea plants 25 days post-inoculation with R. leguminosarum.  Plants were removed from the vermiculite-sand mixture, placed on ice, and nodules were picked from the roots and immediately flash frozen in liquid nitrogen.  Each biological replicate consisted of nodules from three plants from a single Leonard assembly, and enough nodules were collected to fill a 2 mL centrifuge tube to approximately the 500 µL mark.  Three biological replicates were prepared per strain, and nodules were stored at -80°C until use.  For RNA isolation, all nodules per sample were placed in an autoclaved mortar together with 800 µL of 1% β-mercatoethanol in nuclease-free water (Bioshop) and immediately crushed using an autoclaved pestle.  Approximately 500 µL of crushed nodule solution was transferred to a 1.5 mL centrifuge tube on ice, and mixed with 500 µL of room temperature hot phenol solution (per 6 mL: 5 mL of hot phenol buffer [20 mM Tris-HCl pH 7.5, 400 mM NaCl, 40 mM EDTA, 1% SDS, in nuclease-free ddH2O] and 1 mL unbuffered phenol).  The mixtures were vigorously vortexed for 20 seconds then immediately incubated in a water bath at 95°C for 1 minute.  Samples were centrifuged at 16,000 g for 10 minutes at 4°C, and the supernatant was transferred to a fresh 1.5 mL nuclease-free centrifuge tube.  The supernatants were treated with two rounds of unbuffered-phenol:chloroform (1:1) extraction, followed by one round of chloroform extraction.  The nucleic acids were then precipitated by mixing the supernatant with one-tenth sample volume of 3 M sodium acetate and 2x sample volume of isopropanol, mixing, and the mixture was incubated on ice for 30 minutes.  The nucleic acids were pelleted by centrifugation at 16,000 g for 10 minutes, the pellet washed with ice cold 70% ethanol, and air dried.  The pellet was resuspended in 80 µL of nuclease-free ddH2O, mixed with 10 µL DNase I buffer and 10 µL DNase I (20 units; NEB catalog # M0303S), and incubated for 20 minutes at room temperature.  The 100 µL sample was then transferred to a fresh 1.5 mL nuclease-free centrifuge tube, extracted once with unbuffered-phenol:chloroform (1:1) and once with chloroform, and the RNA precipitated as before.  Each RNA pellet was then resuspended in 100 µL water.  A second round of DNase I treatment, extraction, and RNA precipitation was performed as described above.  The final RNA pellets were each resuspended in 100 µL of water, the integrity of the RNA confirmed with PCR and MOPS-formaldehyde gels (and later with BioAnalyzer), and the remainder of the samples stored at -80°C until further use.
RNA libraries were prepared for sequencing using standard Illumina protocols by the Farncombe Family Digestive Health Research Institute located at McMaster University (Hamilton, Ontario, Canada).  Depletion of the plant and bacterial rRNA was performed using Illumina ribo-zero kits with a 50:50 mixture of plant and bacteria probes.

treatment_protocol: Plants were inoculated with either a wild type dctA+ Rhizobium leguminosarum bv. viciae 3841 bacterial strain, or a R. leguminosarum bv. viciae 3841 derivative that was dctA- and expressed the maeP gene of Streptococcus gallolyticus in trans.

tag: plant cultivar

value: Homesteader

tag: agent

value: Rhizobium leguminosarum bv viciae 3841

tag: plant genotype

value: Wild type

tag: bacterial strain

tag: bacterial genotype

value: dctA- maeP+

tag: plant growth condition

value: Nitrogen free


title: Unstimulated_rep2

tag: cell type

value: derived cardiomyocytes from hiPSC


title: Nodule_dctA-_maeP_2


title: Nodule_dctA-_maeP_3


title: 5644_LexAO_DIM5_K9me3_21617

description: Tethered DIM-5 with LexAO binding site integrated at his-3.

tag: strain

value: his-3+::LexAO; DIM-5-LexADBD::hph


title: 5649_LexAO_K9me3_21617

description: LexAO binding site integrated at his-3.


title: Nodule_wild_type_3

value: dctA+ maeP- (wild type)


title: Nodule_wild_type_2


title: Nodule_wild_type_1


title: Endothelin-1 + Importazole_rep4

tag: treatment

value: 10 nM Endothelin-1 + 10 uM Importazole


title: Repeat treated Timepoint 16

status: Public on Sep 15 2017

city: East Lansing

first: George

last: Mias

address: 775 Woodlot Drive, Rm1319

department: Biochemistry and Molecular Biology

institute: Michigan State University

data_processing:
Data QC using FastQC.  150bp long reads were trimmed 15bp from both ends. 125bp long reads were trimmed 12bp from the left and 10bp from the right. 100bp long reads were trimmed 12bp from the left and 15bp from the right.
Trimmed paired reads mapped using Tophat 2.1.0 (using --no-coverage-search --library-type fr-firststrand)
Mapped files quantitated using Cufflinks (--no-effective-length-correction, using hg38 annotation)
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample corresponding to Cufflinks output.

source_name: B-Lymphoblastoid Cell Line

growth_protocol: A BLCL line (Donor 165/Astarte Biologics) cells were grown to a a density of 0.6-0.8 x 10^6/mL in 15% fetal bovine serum and 1% antibiotic-antimycotic.

extract_protocol:
For RNA-Seq, total RNA was extracted using the Qiagen RNeasy kit (Qiagen) and treated with RNAse-free DNase (Qiagen). RNA from ~2 million cells was extracted, with concentration measured on a Qubit (Life Technologies). Additionally, RNA integrity was checked on a Bioanalyzer (Agilent, Santa Clara CA) and only RNA with an RNA integrity number (RIN) of > 7  was used for subsequent library construction.
Libraries were constructed using Illumina's TruSeq kits with Ribo-Zero following the manufacturer's protocol. cDNA was quantitated on a Qubit (Life Technologies) and additionally the library quality was evaluated on a Bioanalyzer (Agilent). Libraries  for the repeat experiment were constructed through Novogene, also using Illumina protocols.

treatment_protocol: In the first experiment, cells were grown together and separated into two flasks at the beginning of the time course (corresponding to a “treated” and an “untreated” condition). Both flasks were sampled simultaneously every half hour over 12 hours (24 time points). The difference between the conditions occurs at time point T7 where, after taking a sample, the cells corresponding to the “treated” condition were treated with Everolimus at a concentration of 50nM.  In the validation repeat experiment, cells were grown under the same conditions and separated into two flasks at the start of the timecourse experiment again corresponding to a “treated and “untreated” condition. The validation sampled time points 0, 7, 10, 13, 16, and 22.

tag: cell line


title: Endothelin-1 + Verapamil_rep4

value: 10 nM Endothelin-1 + 1 uM Verapamil


title: Repeat treated Timepoint 10


title: Repeat treated Timepoint 13


title: Endothelin-1 + Verapamil_rep1


title: Endothelin-1 + Vehicle_rep2

value: 10 nM Endothelin-1 + Vehicle (0.1% DMSO)


title: Repeat treated Timepoint 7

description: treatment after sampling


title: Repeat treated Timepoint 0


title: Repeat Untreated Timepoint 16


title: Repeat Untreated Timepoint 22


title: Repeat Untreated Timepoint 10


title: Repeat Untreated Timepoint 13


title: Eyeless-Gal4,UAS-Dl, RNAi 37547_3

city: Leuven

first: Sofie

last: Demeyer

address: Herestraat 49 / bus 912

department: Center for Cancer Biology

country: Belgium

institute: KULeuven / VIB

data_processing:
cleaning .fastq files with fastq-mcf, quality control with fastQC
mapping to genome with TopHat2
count reads with htseq-count
Genome_build: Drosophila Melanogaster (dm6), Homo Sapiens (GRCh37/hg19)
Supplementary_files_format_and_content: tab-delimited text files contain raw mRNA counts

source_name: eye-antennal imaginal discs

extract_protocol:
Maxwell 16 LEV simplyRNA Cells Kit according to manufacturers instructions (Promega)
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: condition

value: Dl overexpression + Not3 downregulation

tag: tissue

value: eye-antennal imaginal discs

tag: genotype/variation

value: Sensitized + UAS RNAi Not3 37547v


title: Eyeless-Gal4,UAS-Dl, RNAi 37545_3

value: Sensitized + UAS RNAi Not3 37545v


title: Eyeless-Gal4,UAS-Dl, Not3P_2

value: Sensitized + Not3-P element


title: Eyeless-Gal4,UAS-Dl, Not3P_3


title: Eyeless-Gal4,UAS-Dl, RNAi 37547_2


title: Eyeless-Gal4,UAS-Dl, RNAi 37545_2


title: Repeat treated Timepoint 22


title: Eyeless-Gal4,UAS-Dl, RNAi 37547_1


title: Eyeless-Gal4,UAS-Dl, RNAi control_3

value: Dl overexpression + white downregulation

value: Sensitized + UAS-RNAi w


title: Eyeless-Gal4,UAS-Dl, RNAi 37545_1


title: Repeat Untreated Timepoint 0


title: Eyeless-Gal4,UAS-Dl, RNAi 105990_3

value: Sensitized + UAS RNAi Not3 105990v


title: Eyeless-Gal4,UAS-Dl, RNAi control_1


title: Eyeless-Gal4,UAS-Dl, RNAi control_2


title: Repeat Untreated Timepoint 7


title: Eyeless-Gal4,UAS-Dl, RNAi 105990_1


title: Eyeless-Gal4,UAS-Dl, RNAi 105990_2


title: Eyeless-Gal4, UAS-Dl parental_3

value: Dl overexpression

value: Sensitized parental


title: Eyeless-Gal4, UAS-Dl parental_2


title: Eyeless-Gal4, UAS-Dl parental_1


title: JURKAT_siRNA3_4h

source_name: JURKAT cell line

value: 4h after Actinomycin D

value: JURKAT

value: CNOT3 knockdown with si328


title: JURKAT_siRNA2_4h


title: JURKAT_siRNA1_4h


title: JURKAT_Ctrl3_4h

value: control sample


title: JURKAT_Ctrl2_4h


title: JURKAT_Ctrl1_4h


title: Eyeless-Gal4,UAS-Dl, Not3P_1


title: cdk9-G57S homozygous mutant

city: Evanston

first: Richard

last: Carthew

state: IL

address: 2205 Tech Drive

institute: Northwestern University

data_processing:
Bowtie
Genome_build: dm3

source_name: whole larvae

growth_protocol: grown at 25˚C

extract_protocol:
Trizol
Illumina

value: whole larvae

tag: age

value: 48 hour

tag: genotype

value: cdk9-G57S homozygous mutant


title: Wildtype control

value: wild type


title: JURKAT_siRNA2_2h

value: 2h after Actinomycin D