title: ddx39a_24hpf_RIPseq


title: CN3

status: Public on Mar 27 2017

type: SRA

city: Moscow

first: Anastasia

last: Nikitina

address: Malaya Pirogovskaya

department: Department of Molecular Biology and Genetics

country: Russia

institute: Federal Research and Clinical Center of Physical-Chemical Medicine of FMBA of Russia

library_source: transcriptomic

data_processing:
Basecalling was performed by Torrent Suite 5.0 fastqCreator v3.4.56313 BaseCaller --barcode-filter 0.01 --barcode-filter-minreads 10 --phasing-residual-filter=20 -max-phasing-levels 2
Reads filtering was carried out by Cutadapt -q 15 -m 25
Reads were then mapped to human genome version hg19 using STAR.
Genomic coordinates of genes encoding ribosomal RNAs were combined in one .bed file and intersectBed was used to remove from .bam files all aligned reads corresponding to these genes.
HTSeq was used to count the number of reads mapping to a particular gene.
Genome_build: hg19 Gencode Release 19 (GRCh37.p13)
Supplementary_files_format_and_content: *txt: Each tab-delimited text file includes raw read counts corresponding to a particular gene represented by its Ensembl ID (Release 19 (GRCh37.p13)) for a given sample.

molecule: total RNA

organism: Homo sapiens

source_name: normal prostate tissue, PCa patient

extract_protocol:
AllPrep DNA/RNA FFPE Kit (Qiagen) and RNeasy FFPE kit (Qiagen) were used according to manufacturer's instructions to extract RNA from FFPE samples. DNA contaminations were removed with the use of DNase I (Fermentas) following the manufacturer's recommendations.
Transcriptome libraries were constructed using Ion Total RNA-seq Kit v2 (Life Technologies) with the following modifications of the protocol. For RNA fragmentation, 1 ul of 10x RNase III buffer (Life Technologies) was added to 9 ul of RNA solution and heated for 10 min at 95 ˚С followed by immediate snap-cooling on ice. After that, 1 ul of 10 uM ATP and 1 ul of polynucleotide kinase (Fermentas) were added to the solution from the previous step and the whole mix was incubated at 37˚С for 30 min. The fragmented RNA was cleaned up using Micro Bio-Spin Chromatography Columns (Bio-Rad). Further steps of library preparation including adapter ligation, first-strand cDNA synthesis and amplification were carried out in accordance with the manufacturer's instructions.

tag: diagnosis

value: prostate cancer

tag: tissue

value: normal prostate tissue

tag: age at operation

tag: psa level (ng/ml)

tag: gleason sum

tag: primary gleason score

tag: secondary gleason score

tag: extraprostatic invasion

value: no

tag: population

value: Russian

tag: patient identifier


title: CN13


title: CP3

source_name: tumorous prostate tissue, PCa patient

value: tumorous prostate tissue


title: CN1


title: CN11


title: HLF330_day21

city: Minneapolis

first: Ying

last: Zhang

state: MN

address: 117 Pleasant St S.E.

department: Minnesota Supercomputing Institute

country: USA

institute: University of Minnesota

description: Sample name: DGT024

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequence quality was accessed via FastQC, and none of the preprocessing (trimming, filtering or masking) is necessary.
Raw paired-reads were mapped to human genome (hg19) using Tophat 2.0.8 following parameters: --segment-mismatches 1.
Raw counts of reads uniquely mapped to each gene in the hg19 UCSC annotation were calculated using R package GenomeRanges.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include log2 transformed counts of reads per gene for each Sample

source_name: Primary fibroblasts from control lung tissue

growth_protocol: Lung tissue was minced and outgrowth cells were collected like normal at or before p4 (DMEM + 10% FBS, 37°C, 10% CO2)

extract_protocol:
Freshly sorted SSEA4þ/SSEA1/small cells or their progeny (IPF and control, n Z 4; ie, 16 samples total) and were harvested and RNA was isolated using Trireagent. A minimum of 1 µg of total RNA per sample was used for the construction of sequencing libraries.
Steps include oligo-dT purification of polyadenylated RNA, and reverse trascription to create cDNA. The cDNA is fragmented, blunt-ended, and ligated to indexed (barcoded) adaptors. The library was size selected (in the case of paired-end RNA-Seq), size distribution validated using capillary electrophoresis, and quantified using fluorimetry (PicoGreen) and via Q-PCR.

treatment_protocol: The cells were labeled with SSEA4, and FACS sorted for SSEA4 and FSC/SSC.    For the 21 day progeny collection, 100 of the small SSEA4+ cells were cultured in 12 well plate wells for 21 days.

value: lung

tag: cell type

value: fibroblast

tag: disease state

value: control

tag: harvest stage

value: Day 21


title: mock induced THP-1 cells 2

city: Shanghai

first: Shanshan

last: Zhu

address: 320 Yue-Yang Road

country: China

institute: CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

description:
mock induced THP-1 cells
211

data_processing:
cleaned RNA-Seq reads from BGI
map to hg19 reference genome using tophat2 (v2.0.12) with parameters “-g 1 -G knowGene.gtf -m 2”
count reads for each gene using samtools (0.1.18-dev)
calculate normalized signal for each gene as RPKM by considering total mapped reads
Genome_build: hg19
Supplementary_files_format_and_content: gene expression matrix

source_name: peripheral blood

extract_protocol:
Total RNAs from TPA induced or mock induced THP-1 cells were prepared using miRNeasy mini kit (Qiagen) with on-column DNase I digestion, followed by reversed transcription and sequencing using Illumina platform by Beijing Genomics Institute (BGI)
Library construction was done by Beijing Genomics Institute (BGI), according to standard Illumina RNA-Seq protocol

tag: cell line

tag: treatment

value: mock

tag: orginal tissue

value: human peripheral blood


title: mock induced THP-1 cells 1

description:
mock induced THP-1 cells
210


title: TPA induced THP-1 cells 2

description:
TPA induced THP-1 cells
213

value: TPA


title: HLF330_day0

description: Sample name: DGT023

value: Day 0


title: TPA induced THP-1 cells 1

description:
TPA induced THP-1 cells
212


title: IPF332_day21

description: Sample name: DGT020

source_name: Primary fibroblasts from IPF lung tissue

value: Idiopathic Pulmonary Fibrosis (IPF)


title: IPF329_day21

description: Sample name: DGT018


title: IPF329_day0

description: Sample name: DGT017


title: IPF332_day0

description: Sample name: DGT019


title: HLF321_day21

description: Sample name: DGT016


title: HLF311_day21

description: Sample name: DGT015


title: IPF293_day21

description: Sample name: DGT012


title: IPF292_day21

description: Sample name: DGT011


title: IPF292_day0

description: Sample name: DGT005


title: HLF311_day0

description: Sample name: DGT009


title: CP1


title: RNA-Seq Wt1

city: Midlothian

first: Peter

last: Hohenstein

address: Easter Bush Campus

department: The Roslin Institute

country: United Kingdom

institute: University of Edinburgh

description: Processed data file: MDA157_untreated_transcriptome_significant_regulation.txt

data_processing:
Reads were aligned to hg19 using TopHat2 (v2.0.3) Kim et al. Genome Biology 14:R36 (2013).
Differential expression was called using Cufflinks (2.0.1) Trapnell et al. Nature Protocols 7, 562–578 (2012) and by DESeq2 Love et al. Genome Biology 15:550 (2014).
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: MDA157_untreated_transcriptome_significant_regulation.txt: Tab-delimited text file includes log2 fold change in gene expression.

source_name: MDA-MB-157 cell line, Wt1 KO

extract_protocol:
Total RNA was extracted using RNeasy Mini Kit (Qiagen) with on-column DNase digestion as per manufacturer’s instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: breast cancer cell line

value: Wt1 KO


title: HLF271_day0

description: Sample name: DGT008


title: RNA-Seq lacZ

source_name: MDA-MB-157 cell line, lacZ

value: lacZ


title: HLF271_day21

description: Sample name: DGT014


title: VM_9w_a

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

description: Sample name: Embryo1772-078-001

data_processing:
Read processing was performed as described (Islam et al., Nat Methods. 2014 Feb;11(2):163-6), except that read counts were kept and molecule counts disregarded.
The first 6 bases of each read represent a random Unique Molecular Identifier (UMI) that might be used for molecule counting of genes with low abundance that do not saturate UMIs. After follows three or more Gs, stemming from the template switching at the mRNA 5' end during first strand cDNA sythesis.
Genome_build: UCSC hg19
Supplementary_files_format_and_content: Tab-delimited table of read counts

source_name: ventral midbrain

extract_protocol: Human fetal tissues were collected from routine termination of pregnancies at Addenbrooke’s Hospital (Cambridge) and dissected in HIBERNATE media. Ethical approval for the use of postmortem human fetal tissue was provided by the National Research Ethics Service Committee East of England - Cambridge Central (Local Research Ethics Committee, reference no. 96/085).  Tissue pieces were dissociated using Papain Dissociation System (Worthington) following the manufacturer’s recommendations, adjusting incubation time based on tissue piece size, 25-45 min. Glass pipettes of increasingly smaller tip diameter were used to dissociate to single-cell suspension followed by a centrifugation through a BSA single step discontinuous density gradient. Then cells were filtered with 20 μm strainer (Partec CellTrics). Cells were pelleted, resuspended, and stored in N2 media with DNaseI until use. Approximately 800 cells were used from each sample.

value: ventral midbrain

tag: age

value: 9 week embryo


title: hIA_d

description: Sample name: AstroDiffC10

source_name: cell line

growth_protocol: SVGp12 astrocytes (ATCC cat. n CRL86-21), were initially grown according to manufacturer’s instructions to a maximum of passage 3.

extract_protocol: 600-800 cells were lysed and processed further for library preparation according to the protocol in Zeisel et al. (Science 347: 1138–42, 2015)

treatment_protocol: Tet-O-FUW-miR218, Tet-O-FUW-Ascl1, Tet-O-FUW-Lmx1a, and Tet-O-FUW-NeuroD1 lentiviruses were produced in HEK293FT packaging cells (Invitrogen). Cells were infected with viruses in their normal culture medium supplemented with 4μg/ml polybrene overnight. The cells were switched to fresh medium that was replaced every 2 days. After 5 days of amplification the expression of the transgenes was switched on by addition of doxycycline (2μg/mL, Sigma). This doxycycline-supplemented medium was changed every other day until day the end of the experiment. After 48h the medium was changed to KON3 (KODMEM, KSR 15%, 0,25% penicillin/streptomycin (Invitrogen), 25μg/ml insulin, 50μg/ml transferrin, 30nM sodium selenite, 20nM progesterone, 100nM putrescine (Sigma)) containing doxycycline, CT99021 (1μM, Tocris) and Shh (200ng/mL, R&D). This medium was changed until a total of 10 days doxycycline treatment.

value: SVGp12 +NEUROD1+ASCL1+LMX1A+mir218


title: iDA_a

description: Sample name: AstroDiffB0


title: iDA_b

description: Sample name: AstroDiffD0


title: iDA_c

description: Sample name: AstroDiffB10


title: VM_7w_b

description: Sample name: Embryo1772-122-223

value: 7 week embryo


title: hIA_b

description: Sample name: AstroDiffC0

growth_protocol: SVGp12 astrocytes (ATCC cat. N CRL86-21), were initially grown according to manufacturer’s instructions to a maximum of passage 3.


title: VM_9w_b

description: Sample name: Embryo1772-078-107


title: VM_7w_a

description: Sample name: Embryo1772-071-029


title: HLF321_day0

description: Sample name: DGT010


title: hIA_c

description: Sample name: AstroDiffA10


title: non-AMSinHighAltitude rep5

city: Chongqing

first: bao

last: liu

address: 30 Gaotanyan street

department: Institute of Medicine and Hygienic Equipment for High Altitude Region

institute: 3rd Military Medical University

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19 whole genome using  TopHat v2.1.0
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated.
Identify the differentially expressed transcripts of individuals before and after exposure to high altitude by using density-based pruning algorithm.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

source_name: Peripheral venous blood

extract_protocol:
Peripheral venous blood was sampled, flash frozen on ice and followed by -80 ℃, and RNA was harvested using PAXgene Blood RNA Kit. Illumina TruSeq RNA Sample Prep Kit v2 was used with 200ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: ethnicity

value: Chinese Han

tag: phenotype

value: non-AMS

value: Peripheral venous blood

tag: altitude

value: high


title: non-AMSinHighAltitude rep3

tag: tissue


title: non-AMSinHighAltitude rep4


title: non-AMSinHighAltitude rep1


title: non-AMSinPlain rep5

value: low


title: non-AMSinPlain rep4


title: IPF293_day0

description: Sample name: DGT006

value: lung

value: fibroblast


title: non-AMSinPlain rep3


title: non-AMSinPlain rep1


title: AMSinHighAltitude rep5

value: AMS


title: RNA-Seq untreated

source_name: MDA-MB-157 cell line, untreated

value: untreated


title: AMSinHighAltitude rep4


title: iDA_d

description: Sample name: AstroDiffD10


title: AMSinHighAltitude rep3


title: hIA_a

description: Sample name: AstroDiffA0


title: AMSinPlain rep5


title: AMSinPlain rep4


title: non-AMSinHighAltitude rep2


title: AMSinPlain rep2


title: AMSinPlain rep1


title: AMSinPlain rep3


title: A70_Q113

last: Jin

address: 320 Yue Yang Road

department: Institute of Health Sciences

institute: Shanghai JiaoTong University

data_processing:
Basecalls performed using CASAVA version 1.7
Sequences are aligned with tophat 2.0
Reads mapped to multiple position are removed.
Only samples with unique reads > 0.5 million were used.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were called using cufflinks 2.2.1.
Only transcripts with length>500bp were retained.
The values of Log2(RPKM+1) were calculated and represented.
Genome_build: mm10 (from Illumina iGenome)
Supplementary_files_format_and_content: Tab-delimited text file

molecule: polyA RNA

organism: Mus musculus

source_name: Embryo

extract_protocol:
Embryos were collected in the M15 medium. One single embryo was used at a time, which was incubated in EDTA-PBS, and then treated by Trypsin. The parietal endoderm and ectoplacental cone were easily removed, and the remaining embryo was transferred into the MEM medium with 10% FBS before dissociating into single-cell suspension. The single cells were picked into a lysate buffer by a glass pipette assembled on a micromanipulator, followed by reverse transcription directly on the whole-cell lysate. For cells from E5.5 and E6.5_Early embryos (I-III), and cells from E6.5_Late embryo (X), cDNAs were prepared according to previous methods. Briefly, oligo (dT) primers, which were complementary to poly (A) tails of mRNAs, were incorporated into the 5' end of the first-strand cDNAs during the reverse transcription step. And then a poly (A) tail was added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. After the second strands were synthesized, cDNAs were amplified by 20+12 cycles of PCR. For cells from embryos E6.5_Late (VII) and E6.5_Late (VIII), mRNAs were reverse transcribed by SMARTScribeTM Reverse Transcriptase and then directly amplified by 18+12 cycles. Amplified cDNAs were first examined for the expression of a few marker genes (Oct4, Hand1, Gata6 or a combination of germ-layer markers) by quantitative real-time PCR. cDNA from selected cells were purified either by gel electrophoresis or Agencourt AMPure XP beads. For samples purified by gel electrophoresis, only transcripts with length > 500 bp were cut from the gel. For samples purified with Agencourt AMPure XP beads, 80% ethonal was used so that the transcripts recovered are mainly >500bp.
cDNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain

value: Late preMEN

tag: developmental stage


title: A83_Q32

value: ME


title: A89_Q67


title: A82_Q30


title: A96_Q89

value: ME(EXEM)


title: A63_Q46

value: Other


title: IRF4_BATFKO

city: Chiba-shi

first: Arifumi

last: Iwata

state: Chiba

address: 1-8-1 Inohana, Chuou-ku

department: Department of Allergy and Clinical Immunology

country: Japan

institute: Chiba University

library_source: genomic

data_processing:
Basecalls performed using CASAVA
Reads were alignted using Bowtie 1.1.1
Uniquely mapped reads were masked using samtools with blacklist of ENCODE project and RepeatMasker in the UCSC genome browser.
For ChIP-seq, duplicated tags were discarded by Homer.  For ChIP-exo, duplicated tags were kept and 50 bp of tags were shrunken to first 1 bp of 5' position.
For ChIP-seq, bedgraph were made using makeUCSCfile program of the Homer software package.  For ChIP-exo, bedgraph were made using genomecov program of the bedtools software package.
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph

molecule: genomic DNA

source_name: cultured TH2 cells

growth_protocol: For samples of secondary stimulation, CD4 T cells were activated on anti-CD3e and anti-CD28 coated plates under TH2conditions (anti-IFN-g,, anti-IL12, IL-4. On day 3, cells were diluted three-fold in fresh media on uncoated plates. On day 7, cells were re-stimulated under the same conditions. Cells were prepared for ChIP after PMA/ionomycin stimulation on day 4 of secondary stimulation. For samples of primary stimulation, naive CD4 T cells were activated with soluble anti-CD3e at various concentrations and anti-CD28 cross linked by plate-bound anti-hamster IgG under TH2 conditions.

extract_protocol:
Cells were crosslinked with 1% formaldehyde for 8 min at room temperature, quenched with 1.25M Glycine, twice washed with PBS.  Chromatin was sonicated for shearing DNA size from 140 to 300 bp. Chromatin fragments were immunoprecipitated overnight at 4 °C with Dynabeads Protein A or G bound to anti-BATF or anti-IRF-4 (sc-6059x, Santa Cruz Biotechnology).  For ChIP-seq, ChIPed DNA was eluted and reverse-crosslinked for 6 hr. DNA was purified with phenol:chloroform extraction followed by ethanol precipitation. For ChIP-exo, see library construction protocol.
For ChIP-seq, libraries were prepared using ThruPLEX-FD kit (Rubicon Genomics).  For ChIP-exo,  ChIPed DNA were performed by the following enzymatic reactions; End polishing, ligation of P7 exo-adaptor, nick repair, exonuclease reaction. DNA was eluted and reverse crosslinked. The libraries were generated by P7 primer extension, ligation of P5 exo-adaptor, PCR amplification for 12 cycles and were cleaned up by AMPure XP.  All libraries were sequenced using Illumina HiSeq 2500 as single reads extending 50 bases.

tag: method

value: ChIP-seq

value: BATFKO, secondary stimulation

tag: chip antibody


title: IRF4_WT_rep1

value: WT, secondary stimulation


title: Q77_122


title: Q68_126


title: BATF3_BATFKO


title: MGHS72h_1

city: Geneva

first: Celine

last: Delucinge

address: 1 rue Michel Servet

department: iGE3 Genomics Platform

country: Switzerland

institute: University of Geneva

description: Processed data file: Microglia_normcount_table.txt

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence with FASTX-toolkit, then mapped to mm10 whole genome using TopHat v2.0.9.
Count data are prepared with HTSeq v.0.5.4p3.
Normalization and differential expression analysis performed with R/Bioconductor edgeR package v.3.3.6.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Microglia_normcount_table.txt: Tab-delimited text file includes normalized counts for each sample.

source_name: microglia, hemi-section, 72hr

extract_protocol:
RNeasy Mini Kit (Qiagen, Maryland, USA), including DNase treatment.
Clontech Smarter Ultra low + Illumina Nextera library protocols.

tag: genotype/variation

tag: injury

value: hemi-section

tag: time

value: 72 hours after injury

value: microglia


title: non-AMSinPlain rep2

value: Chinese Han

value: non-AMS

value: Peripheral venous blood


title: A84_Q34


title: MGNI_1

source_name: microglia, non-injured

value: non-injured

value: start point

tag: cell type


title: MGNI_3


title: MGHS72h_3


title: MGNI_2


title: MGHS2w_3

source_name: microglia, hemi-section, 2wk

value: 2 weeks after injury


title: MGHS72h_2


title: AMSinHighAltitude rep1

value: AMS

value: high


title: MGHS2w_1


title: MGHS2w_2


title: MGHS1w_2

source_name: microglia, hemi-section, 1wk

value: 1 week after injury


title: MGFT72h_3

source_name: microglia, full transection, 72hr

value: full transection


title: MGFT72h_2


title: MGFT72h_1


title: MGHS1w_1


title: MGFT2w_3

source_name: microglia, full transection, 2wk


title: MGFT2w_2


title: MGFT1w_1

source_name: microglia, full transection, 1wk


title: MGFT1w_3


title: MGFT1w_2


title: A133

value: Late preMEN


title: A135

value: ME


title: A138


title: A132


title: A128

value: the rest of Late EPI


title: A134


title: A130

value: ME(EXEM)


title: A131

value: Other(EXEM)


title: A113_n68

value: VE


title: A115_n83


title: A110_n33


title: A129

value: EXE-like


title: A111_n40

value: EXE


title: A109_n32


title: A112_n47


title: A108_n31


title: A107_n12


title: Q69_121

value: DE


title: Q49_125

value: Other


title: A106_Q162


title: Q38_124


title: A105_Q156


title: AMSinHighAltitude rep2

value: Chinese Han

value: Peripheral venous blood


title: A104_Q140


title: A101_Q124


title: A103_Q139


title: A100_Q119


title: A114_n82


title: A94_Q80


title: A99_Q103


title: A92_Q72


title: A91_Q71


title: MGFT2w_1

value: 2 weeks after injury

value: microglia


title: A88_Q57


title: A86_Q47


title: A81_Q28


title: A85_Q41


title: Q95_127


title: A77_Q6


title: A78_Q16


title: A76_Q4


title: A80_Q19


title: A71_Q122


title: A69_Q110


title: A102_Q138


title: A66_Q56


title: A68_Q92


title: A62_Q43


title: A65_Q54


title: A98_Q96


title: A97_Q93


title: A120_b115


title: A61_Q40


title: A60_b141


title: A59_b140


title: A57_b135


title: A58_b137


title: A56_b131


title: A55_b130


title: A54_b129


title: A51_b110


title: A48_b98


title: A50_b109


title: A47_b96


title: A52_b119


title: A46_b92


title: A43_b70


title: A45_b80


title: A40_b60


title: A67_Q58


title: A41_b62


title: A39_b59


title: A37_b43


title: A38_b55


title: A42_b69


title: A36_b41


title: A35_b38


title: A32_b31


title: A34_b36


title: A33_b33


title: A53_b127


title: A29_b16


title: A28_b15


title: A79_Q17


title: A27_b8


title: A30_b22


title: A19_b139


title: A18_b134


title: A15_b123


title: A16_b125


title: A14_b116


title: A13_b101


title: A11_b85


title: A12_b88


title: A10_b84


title: A9_b77


title: A7_b65


title: A6_b49


title: A8_b71


title: A4_b6


title: A118_a54


title: A26_a125


title: A117_na47


title: A25_a114


title: A31_b26


title: A119_a94


title: A23_a91

value: VE-like


title: A21_a34


title: A20_a27


title: A22_a59


title: S12D122

value: the rest of Early EPI


title: A1_a10


title: A3_a124


title: S12D043


title: S12D125


title: S12D124


title: A2_a40


title: S13D036


title: S12D033


title: S12D064


title: S12D038

value: Early preMEN


title: S13D032


title: S12D010


title: A5_b28


title: Input_DKO

description: input

value: cultured TH2 cells

tag: genotype

tag: stimulation

value: secondary stimulation

value: no antibody


title: Input_WT

description: input

value: WT


title: exo_BATF_rep4

description: ChIP-exo

value: anti-BATF


title: exo_IRF4_rep1

description: ChIP-exo


title: exo_BATF_rep2

description: ChIP-exo


title: exo_BATF_rep3

description: ChIP-exo


title: exo_IRF4_rep2

description: ChIP-exo


title: IRF4_2ng

description: ChIP-seq

value: primary stimulation, day 4, 2.2ng


title: IRF4_10ng

description: ChIP-seq

value: primary stimulation, day 4, 10ng


title: IRF4_day2

description: ChIP-seq

value: primary stimulation, day 2, 10ng


title: IRF4_DKO

description: ChIP-seq


title: IRF4_WT

description: ChIP-seq


title: BATF_WT

description: ChIP-seq


title: Control 3

city: Washington

first: Ray-Chang

last: Wu

state: DC

address: 2300 I St, NW

department: Biochemistry and Molecular Medicine

institute: George Washington University

data_processing:
After sequencing, the analysis was performed using EA | Q² Solution's in-house developed RNAv9 pipeline which uses a variety of internally developed and open source programs made available at https://code.google.com/p/ea-utils/.
Any read that has one particular base at ≥ 95% frequency (homopolymer filtering), or has more than 4 Ns within its sequence, or has an average Q score below 25, or has sa equence corresponding to a Illumina adapters, or are shorter than 25 nt in length, was removed.
STAR version 2.4 was used to align the filtered reads to the Ensembl mouse transcriptome.
Genome_build: mm10
Supplementary_files_format_and_content: Comparison-Genes.txt
Supplementary_files_format_and_content: Field_Descriptions.txt contains descriptions of column headers in Comparison-Genes.txt

source_name: Testes, control

growth_protocol: standard chow

extract_protocol:
Total RNA was purified using an RNeasy plus kit (Qiagen), and was treated by DNase I (Qiagen).
Samples were prepared and sequenced according to  EA | Q² Solutions standard TS Stranded mRNA sequencing protocol using HiSeq - 2x50bp-PE sequencing on an Illumina sequencing platform.
RNA samples were converted into cDNA libraries using the Illumina TruSeq Stranded mRNA sample preparation kit (Illumina # RS-122-2103), and with dUTP utilized in place of dTTP in the second strand master mix. Forked barcode adaptors were attached to the cDNAs via ligation.
The resulting molecules were PCR amplified. Since only the second strand included the dUTP base, stalling the polymerase, the only viable template was the first strand, thereby preserving the strand information. Final libraries are quantified, normalized and pooled for sequencing.

value: hybrid C57BL6/J and 129/SvEv

value: Arid4b+/Flox; Amh-cre

value: postnatal day 1.5

value: Testes


title: S13D017


title: Control 1


title: Arid4bSCKO 3

source_name: Testes, Arid4bSCKO

value: Arid4b-/Flox; Amh-cre


title: Arid4bSCKO 1


title: Arid4bSCKO 2


title: Control 2


title: S12D066


title: S12D067


title: Cbx3_ChIPseq_NPC_set2

city: Los Angeles

first: Siavash

middle: K

last: Kurdistani

state: CA

address: 615 Charles E Young Dr South

department: Biological Chemistry

institute: UCLA

data_processing:
Reads were mapped to the Mouse (mm9) genome using Bowtie software. Only reads that aligned to a unique position in the genome with no more than two sequence mismatches were retained for further analysis. Duplicate reads that mapped to the same exact location in the genome were counted only once to reduce clonal amplification effects. The genome was tiled with 50 bp windows. Each read was extended by 150 bases (so we refer to tags as the extend read counts within a bin). With custom Python script, the total counts of the input and ChIP samples were normalized to each other and determined enrichment P-value using Poisson distribution.
Genome_build: mm9
Supplementary_files_format_and_content: bed files with ChIP signal.

source_name: Cell line

growth_protocol: V6.5 ESCs were differentiated into NPCs through embryonic body formation for 4 days EBs were dissociated into single cells and plated in tissue culture dish with ITSF media for monolayer NPC generation.

extract_protocol:
Cells were crosslinked with 1% formaldehyde. Cell lysates were obtained from sonicated nuclei and protein-DNA complexes were isolated with indicated antibodies.
Libraries were prepared with KAPA LTP kit according to manufacturer's protocol.

treatment_protocol: Cells cultured in ITSF media were transfected with either control siRNA targeting luciferase or siRNA mediated knockdown of Cbx3 for 72 hrs prior to harvest.

tag: cell line

tag: antibody

value: Anti-Cbx3 antibody, Millipore, 05-690; clone 42s2

value: ESC-derived NPCs

tag: treatment


title: Input_Cbx3_NPC_set2


title: NPC_Med26KD_biological replicate1

data_processing:
Alignment of mRNA-seq reads was performed using default parameters of Tophat version 2.08. Aligned reads were converted to sam format and SAMMate software was used to determine the transcript RPKM (reads per kilobase of exon per million of reads).
Genome_build: mm9
Supplementary_files_format_and_content: csv file with RPKM values

growth_protocol: Undifferentiated V6.5 ESCs were cultured on irradiated MEFs in ESC media: Knockout DMEM (Thermo Fisher) containing 15% FBS, 2000U/ml leukemia inhibiting factor (LIF), 2mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol and penicillin/streptomycin. Monolayer neural differention: ESCs were differentiated into NPCs through embryonic body formation for 4 days EBs were dissociated into single cells and plated in tissue culture dish with ITSF media for monolayer NPC generation.

extract_protocol:
RNAs were extracted using Trizol according to manufacturer's instruction.
Libraries were prepared with an Illumina TruSeq RNA sample preparation kit according to manufacturer's protocol.

treatment_protocol: Transfection of siRNAs was performed on day 5 after the cells were cultured in ITSF media for monolayer NPC generation. RNAs were harvested on day 8, three days after the transfection, and subjected to RNA-seq experiments.

tag: sirna mediated knockdown


title: NPC_Med26KD_biological replicate2


title: NPC_Cbx3KD_biological replicate2


title: NPC_Cbx3KD_biological replicate1


title: NPC_control_biological replicate2

value: Control


title: ESC_Cbx3KD_biological replicate1

value: ESCs


title: Input_Cbx3KD_NPC

source_name: Cell line_input_Cbx3_siRNA

value: none (input)

value: Cbx3 siRNA transfection


title: ESC_control_biological replicate2


title: ESC_control_biological replicate1


title: Pol II_Cbx3KD_NPC

source_name: Cell line_PolII_ChIP_Cbx_siRNA

value: Anti-Pol II antibody, sc-56767


title: IRF4_Batf-HKE

description: ChIP-seq

value: cultured TH2 cells

value: Batf1/3DKO overexpressing Batf-HKE

value: secondary stimulation


title: Input_Control_NPC

source_name: Cell line_input_control_siRNA

value: control siRNA transfection


title: NPC_control_biological replicate1


title: ESC_Cbx3KD_biological replicate2


title: Cdk8_Cbx3KD_NPC

source_name: Cell line_Cdk8_ChIP_Cbx3_siRNA

value: Anti-Cdk8 antibody, sc-1521


title: Cdk8_Control_NPC

source_name: Cell line_Cdk8_ChIP_control_siRNA


title: S12D129

source_name: Embryo

value: the rest of Early EPI


title: Input_Med26_Control_NPC


title: Input_Pol II_NPC

source_name: Cell line_input_no treatment


title: Input_Med26_Cbx3KD_NPC


title: Med26_Cbx3KD_NPC

source_name: Cell line_Med26_ChIP_Cbx3_siRNA

value: Anti-Med26 antibody, sc-48776


title: Cbx3_ChIPseq_NPC

source_name: Cell line_Cbx3_ChIP_no treatment


title: WT_3h

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

institute: Osaka univ.

description: Batf2+/+ BMMφ stimulated with LPS following IFN-γ treatment.

data_processing:
Illumina Casava1.8.2 software used for basecalling.
The low-quality regions of raw reads were trimmed with Btrim (http://graphics.med.yale.edu/trim/readme).
The trimmed reads were mapped onto the reference mouse genome mm9 using TopHat2 in combination with Bowtie and SAMtools.
Gene expression was quantified with Cufflinks.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: Cufflinks_*.txt: Tab-delimited text files include RPKM values.

source_name: LPS-stimulated wild-type BMMφ following IFN-γ treatment

growth_protocol: Bone marrow cells were prepared from the femur and tibia, passed through a nylon mesh, and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 μM 2-mercaptoethanol (2ME), and 10 ng/ml GM-CSF (Pepro Tech, Rocky Hill, NJ). After 6 days, the adherent cells were used as bone marrow-derived macrophages (BMMφ).

extract_protocol:
Total RNA was extracted from BMMφ cells with RNeasy Mini Kit (Qiagen) and processed according to the manufacturer's protocol. The quality of the RNAs was estimated by using the RNA 6000 Nano LabChip Kit (p/n 5065-4476) on the Agilent 2100 Bioanalyzer (G2940BA; Agilent Technologies, Santa Clara, CA).
RNA-sequencing libraries were prepared using a SureSelect Strand Specific RNA Library Preparation kit (Agilent Technologies, Santa Clara, CA). Single-read.

treatment_protocol: Bone marrow-derived macrophages (BMMφ) were stimulated with 100 ng/ml LPS for the indicated periods following 10 ng/ml IFN-γ treatment for 4 h.

tag: strain/background

value: bone marrow-derived macrophages

value: LPS-stimulation following IFN-gamma treatment

tag: time point


title: KO_0h

description: Batf2-/- BMMφ stimulated with LPS following IFN-γ pretreatment.

source_name: LPS-stimulated Batf2-/- BMMφ following IFN-γ pretreatment

value: LPS-stimulation following IFN-gamma pretreatment


title: KO_3h

description: Batf2-/- BMMφ stimulated with LPS following IFN-γ treatment.

source_name: LPS-stimulated Batf2-/- BMMφ following IFN-γ treatment


title: Pol II_Control_NPC

source_name: Cell line_PolII_ChIP_control_siRNA


title: S12D102

tag: tissue

value: VE


title: S12D105

tag: strain


title: S12D104

tag: developmental stage


title: Med26_Control_NPC

source_name: Cell line_Med26_ChIP_control_siRNA

value: ESC-derived NPCs


title: Pol II_ChIPseq_NPC

source_name: Cell line_PolII_ChIP_no treatment


title: S13D043

value: EXE


title: S13D044


title: S12D118


title: S12D128


title: S12D127


title: S12D117


title: S12D123


title: S12D103


title: S12D080


title: S12D116


title: S12D119

value: Early preMEN


title: S12D121


title: S12D120


title: Input_Cbx3_NPC

value: none (input)


title: S12D114


title: S12D115


title: S12D113


title: WT_0h

description: Batf2+/+ BMMφ stimulated with LPS following IFN-γ pretreatment.

source_name: LPS-stimulated wild-type BMMφ following IFN-γ pretreatment


title: S12D111


title: S12D112


title: S12D110


title: S12D109


title: S12D107


title: S12D106


title: S12D098


title: S12D100


title: S12D096


title: S12D094


title: S12D090


title: S12D039


title: S12D092


title: S12D044


title: S12D040


title: S12D091


title: S12D037


title: S12D035


title: S12D036


title: S12D097


title: S12D093


title: S12D034


title: S12D041


title: S12D108


title: S12D029


title: S12D028


title: 40 ScreFF

city: Columbus

first: Paul

middle: T

last: Martin

state: OH

address: 700 Childrens Dr.

department: Center for Gene Therapy

institute: Nationwide Childrens Hospital

data_processing:
Paired-end 150bp sequence reads were generated using the Illumina HiSeq 4000 platform.
Alignment with assembly was done using version 2.5.1b of the RNA-Seq aligner STAR.
Transcript features were identified from the GFF file provided with the GRCh38.p5 assembly from NCBI and raw coverage counts were calculated using HTSeq.
Raw RNA-Seq gene expression data was normalized and post-alignment statistical analyses performed using DESeq2.
Genome_build: GRCm38.p4
Supplementary_files_format_and_content: xlsx file containing abundance measurements and comparison between groups

source_name: Quadriceps muscle

growth_protocol: All mice were maintained in the animal facility of the Research Institute of Nationwide Childrens Hospital following the IACUC guidelines.

extract_protocol:
Trizol extraction of total RNA was performed according to the manufacturer's protocol followed by purification on a silica-gel-based membrane (Rneasy-Mini; Qiagen, Valecia, CA).
Libraries were constructed from first strand cDNA using ScriptSeqTM v2 RNA-Seq library preparation kit. Briefly, 50ng of rRNA-depleted RNA was fragmented and reverse transcribed using random primers containing a 5' tagging sequence, followed by 3'end tagging with a terminal-tagging oligo to yield di-tagged, single-stranded cDNA. Following purification by a magnetic bead-based approach, the di-tagged cDNA was amplified by limit-cycle PCR using primer pairs that anneal to tagging sequences and add adaptor sequences required for sequencing cluster generation. Amplified RNA-seq libraries were purified using AMPure XP System.

tag: age

value: Old (17 months)

value: Pofut1 knockout

value: Quadriceps muscle


title: S12D027


title: 45 ScreFF


title: S12D095


title: S12D042


title: S12D023


title: S12D025


title: S12D024


title: 17 ScreFF


title: exo_BATF_rep1

description: ChIP-exo

source_name: cultured TH2 cells

value: cultured TH2 cells

value: WT

tag: chip antibody

value: anti-BATF


title: S12D032


title: S12D022


title: 83 ScreFF

value: Young (2 months)

tag: genotype/variation


title: S12D021

value: the rest of Early EPI


title: S12D020


title: S12D019


title: 82 ScreFF


title: S12D016


title: 80 ScreFF


title: 34 ScrePlusPlus

value: Skeletal alpha actin cre/Pofut1 wild type


title: S12D015


title: S12D018


title: 35 ScrePlusPlus


title: S12D014


title: S12D012


title: S12D013


title: S12D026


title: 99 ScrePlusPlus


title: S12D011


title: 61 ScrePlusPlus


title: S12D009


title: 94 ScrePlusPlus


title: S12D006


title: S12D089


title: S12D087


title: S12D005


title: S12D083


title: 5 FF

value: Pofut1flox/flox control


title: S12D086


title: 46 FF


title: S12D065


title: S12D061


title: S12D062


title: 92 FF


title: S12D055


title: 32 FF


title: S12D008


title: S12D063


title: 57 FF


title: S12D051


title: S12D007


title: S12D053


title: S12D060


title: S12D050


title: S13D034


title: S12D052


title: S12D056


title: S13D038


title: 93 ScrePlusPlus


title: S13D033


title: S12D088


title: S13D031


title: S13D018


title: S13D023


title: S13D019


title: S13D022


title: S13D025


title: 48 FF


title: S13D016


title: S13D015


title: S13D014


title: S13D013


title: S13D012


title: S13D011


title: S13D009


title: 12h in vivo activated IJ rep 2

first: Marissa

last: Macchietto

address: 117 Pleasant Street SE

institute: University of Minnesota, Minneapolis

description:
PolyA RNA
unstranded, paired-end, read_length= 43 bp

data_processing:
The S. carpocapsae transcriptome (PRJNA202318 downloaded from WormBase ParaSite version WS254) was prepared for indexing using the RSEM command (version 1.2.12) rsem-prepare-reference. The unstranded, paired-end 43 bp RNA-seq reads were mapped to the transcriptome using Bowtie 0.12.8 with the following options: -X 1500 -a -m 200 -S --seedlen 25 -n 2 --offrate 1 -p 64 -v 3. Gene expression was quantified with RSEM, by using the command rsem-calculate-expression. RNA-seq reads were also mapped to the genome using Tophat to compare read mapping percentages between the genome and transcriptome.
Batch correction and normalization of gene expression data: Gene expression counts from RSEM were batch corrected with the R package limma to normalize for technical differences since the 9 hr and 12 hr in vivo activated samples were collected and processed on a different day from other samples. Gene expression in transcripts per million (TPM) was quantile normalized using normalizeQuantiles in R.
Genome_build: S_carpo_v1
Supplementary_files_format_and_content: *isoforms.results.txt: Tab-delimited text files include isoform expression abundances.
Supplementary_files_format_and_content: *genes.results.txt: Tab-delimited text files include gene expression abundances.

organism: Steinernema carpocapsae

source_name: whole individual worm, infective juvenile, in vivo activated, 12h

growth_protocol: Galleria mellonella (waxworms) were purchased from CritterGrub (www.crittergrub.com). Healthy waxworms were sorted to remove wood chips, rinsed with tap water, surface dried on paper towels, and used immediately for infection by IJs or frozen at -80 °C for making waxworm homogenate later. Oregon-R Drosophila flies were kept in standard fly bottles as previously described. Briefly, flies were reared in bottles containing dextrose medium (129.4 g/L dextrose, 7.4 g/L agar, 61.2 g/L corn meal, 32.4 g/L yeast, and 2.7 g/L tegosept; polypropylene round bottom 8oz bottles plugged with bonded dense weave cellulose acetate plugs, Genesee Scientific Cat #49-100) and were housed at 25 °C with 60% relative humidity and a 12 hour light and 12 hour dark cycle. Symbiotic IJs were produced by in vivo culture in waxworms. Twenty clean waxworms were put in a 10 cm Petri dish with a piece of filter paper on the bottom. Symbiotic IJs (~50 IJs/host) of S. carpocapsae (strain All) were suspended in 1 ml of tap water and distributed onto the filter paper. The infection can be scaled up to many plates to obtain more IJs. The infection plates were incubated at 25 °C in the dark for 2 weeks. Then the waxworm cadavers were transferred to White traps (White 1927) in which a piece of 5.5 cm filter paper was raised by the lid of a 3.5 cm Petri dish in a 10 cm Petri dish with a thin layer of tap water (1-2 mm deep). After 1-2 weeks, the IJs gradually migrated into the surrounding water. The IJs were collected and washed with tap water at least 3 times before being stored in tissue culture flasks with vented caps (VWR, 10062-860). Washing a large number of IJs was performed in a glass vacuum filter holder (Fisher Scientific, 09-753-1C) with two layers of 11 µm pore-sized nylon net filters (Millipore, NY1104700). Small populations of IJs were washed in 15 ml tubes by centrifugation at 700 rcf for 1 min at room temperature in a swing-bucket rotor. The supernatant was removed after each wash. The density of IJs was controlled to be lower than 15 IJs/µl to prevent oxygen depletion. The IJs were stored at 15 °C. IJs remain largely quiescent and can usually survive for a year or longer under these conditions. For activation experiments, IJs were used within 3 months after collection. Axenic IJs were produced by in vitro culture on the colonization defective mutant bacterial strain HGB315 of X. nematophila. The mutant bacteria was streaked from a -80 °C glycerol stock onto a NBTA plate (8.0 g/L nutrient agar; 25 mg/L bromothymol blue; 40 mg/L 2,3,5-triphenyltetrazolium chloride) supplemented with 0.1% (w/v) Sodium Pyruvate and a MacConkey plate (Difco MacConkey Agar, #212123) supplemented with 0.1% (w/v) Sodium Pyruvate. All colonies turned dark blue on the NBTA plate and reddish/brown on the McConkey plate (Figure 2¬–figure supplement 3), confirming that they were the phase I bacteria. Mutant X. nematophila was inoculated in 5 ml of Luria-Bertani (LB) broth with 0.1% (w/v) Sodium Pyruvate and cultured overnight at 25-28 °C with shaking at 220 rpm. 100-200 µl of overnight liquid culture was spread on lipid agar plates (8 g/L of nutrient broth, 5 g/L of yeast extract, 2 g/L of MgCl2, 7 ml/L of corn syrup, 4 ml/L of corn oil, and 15 g/L of Bacto Agar) supplemented with 0.1% (w/v) Sodium Pyruvate and incubated at 25-28 °C for 24 hr or until a bacteria lawn formed. 1.5x10^4 symbiotic S. carpocapsae IJs were seeded onto the mutant bacteria lawn. After 3 days, the nematodes developed to mature adults including gravid females, which were washed 3 times in a 15 ml tube with 0.8% NaCl solution. The adult nematodes were treated twice by 15 ml bleach solution (0.5N NaOH and 0.5-0.6% NaClO) for 10 min each time. All the tissues were digested except the eggs/embryos, which were washed 3 times by 0.8% NaCl solution and added to the mutant bacteria lawn on lipid agar plates. Nematodes hatched and developed to adults , which were split to multiple mutant bacteria plates for obtaining more IJs. IJs were washed off the mutant bacteria plates and washed 10 times with 0.8% NaCl solution. 1000 IJs were surface sterilized by 1 ml of 4 mM Hyamine 1622 solution (Sigma, 51126) for 30 min and washed 3 times with 0.8% NaCl solution. The IJs were homogenized in 50 µl of 0.8% NaCl solution by a tissue grinder (Fisher Scientific, 12-141-363) and plated on the LB plates supplemented with 0.1% (w/v) Sodium Pyruvate. The plates were incubated at 28 °C in dark and checked for bacteria growth every day for 3 days. Four replicates were performed for the IJs from mutant bacteria plates and two replicates of 1000 symbiotic IJs were used as the control.

extract_protocol:
A single fully activated nematode was pipetted in a 0.5 µl volume to the side wall of a clear 0.2 ml PCR tube containing 2 µl of Lysis buffer [10 mM Tris-HCl (pH 7.5-8.0), 1% Triton X-100, 1% Tween 20, 0.5 mM EDTA, 3 units of RNase inhibitor (Takara 2313A), 2 mg/ml Proteinase K (VWR, 97062-670)] at the bottom. The nematode was cut by a syringe needle (BD, 305109) into 3-4 fragments when the tube was laid horizontally on the stage of a Leica M80 dissecting microscope. The fragments were immediately spun down into the lysis buffer using a mini centrifuge. The tube was placed on ice for 30-60 min. 8 nematodes were individually processed for each condition, i.e., non-activated, in vitro activated for 12 hrs, in vivo activated for 9, 12, and 15 hrs. The samples were heated to 65°C to denature the protease, and the samples were prepared according to the Smartseq2 protocol (Picelli et al., 2014).
Library preparation was done using the Illumina Nextera tagmentation protocol. Input DNA and reagents were scaled down so that 20ng of cDNA (2.5 ng/ uL) was combined with 12 uL of tagment buffer and 2.1 uL of transposase enzyme in a PCR tube. The tagmentation reaction was carried out for 5 min at 55°C in a thermocycler. The transposase was cleaned up from the DNA using the Qiagen DNA purification kit, and eluted from the Qiagen column with 30 uL of EB. The sample was mixed together with 35 μL of Phusion high fidelity master mix, 2.5 μL 25 μM Nextera adapter ID, and 2.5 μL 25 μM Nextera adapter Ad_noMX in a PCR tube. Samples were spun down quickly, and amplified for 6 cycles using the PCR program with the following settings: 72 °C for 5 min, 98 °C for 30 s, [98 °C for 10 s, 63 °C for 30 s, 72 °C for 1 min] for 6 cycles, 72 °C for 5 min, and hold at 4 °C. PCR primers were cleaned up from the sample by mixing the sample with a 1:1 ratio of Ampure XP beads. Samples were placed on a magnet, and washed with three 200 uL washes of 80% ethanol. The beads were dried for 5 min, and the library was eluted from the beads in 30 uL of elution buffer. Library concentrations were measured with the Qubit fluorometer and Bioanalyzed with the 2100 Agilent Bioanalyzer system. Sample libraries had an average fragment size of 450bp and were sequenced as paired-end 43 bp on the Illumina NextSeq 500 to an average depth of 20 million reads.

treatment_protocol: For in vivo activation of IJs, 10 waxworms were infected by 10,000 IJs in an infection Petri dish as described above. After 0.5 hr of infection, waxworms were transferred to a new Petri dish so that the infection was relatively synchronized. After 9, 12 and 15 hr incubation at 25 °C in dark, the waxworms were dissected in 0.8% NaCl solution. The released nematodes were separated from big tissue debris and washed 3 times with 0.8% NaCl solution in a 15 ml tube. The nematode suspension was placed in a Petri dish and the nematodes (observed with a 10x6 magnification of a Leica M80 dissecting microscope with transmitted light) were individually transferred by a 10 µl pipette to another Petri dish containing 0.8% NaCl solution. This step was repeated two more times to reduce contamination from host debris. For in vitro activation of IJs, 1000 IJs were incubated in 0.085 g of sponge soaked with 1 ml of 25% waxworm homogenate and 1x antibiotics (Sigma, P4083-100ML). After 12 hr incubation at 25 °C in dark, nematodes were washed off the sponge foam and washed in the same way as the in vivo activated nematodes. The activated nematodes were individually pipetted onto a glass slide without a coverslip and examined under a compound microscope to confirm that they have fully expanded pharyngeal bulbs (fully activated).

value: ALL

value: whole body

value: infective juvenile (IJ)

value: in vivo activated


title: 12h in vivo activated IJ rep 1


title: 9h in vivo activated IJ rep 2

source_name: whole individual worm, infective juvenile, in vivo activated, 9h


title: 12h in vivo activated IJ rep 3


title: 12h in vitro activated IJ rep 3

source_name: whole individual worm, infective juvenile, in vitro activated, 12h

value: in vitro activated


title: 12h in vitro activated IJ rep 2


title: 9h in vivo activated IJ rep 3


title: 12h in vitro activated IJ rep 1


title: nonactivated IJ rep 2

source_name: whole individual worm, infective juvenile, nonactivated

value: nonactivated


title: nonactivated IJ rep 3


title: 9h in vivo activated IJ rep 1


title: HWT Start-seq males NextSeq rep1

city: Chapel Hill

first: Michael

last: Meers

state: NC

address: 250 Bell Tower Dr.

institute: UNC Chapel Hill

description:
HWT vs. K36R Start-seq
processed file: obsTSS_calls.full.bed
processed file: nuTSS_calls.full.header.bed

data_processing:
Library strategy: Start-seq (Nechaev et al. 2010, Henriques et al. 2013)
Alignment to reference genome using Tophat2 (RNA-seq) or Bowtie2 (Start-seq, ATAC-seq)
Generation of count tables for RNA-seq using htseq-count
Differential expression analysis of RNA-seq using DESeq2 or Cufflinks
Assignment of nucleotide position for Start-seq peaks using custom scripts
Generation of BigWig files for ATAC-seq samples using Deeptools
Analysis of meta-TSS profiles for ATAC-seq samples using Deeptools
Genome_build: dm3

organism: Drosophila melanogaster

source_name: Whole 3rd Instar Larvae

growth_protocol: 3rd instar larvae were staged by wandering behavior and collected for extraction

extract_protocol:
Total RNA was extracted from isolated nuclei using TRIzol reagent (Thermo Fisher) using manufacturer's protocols.
Start-seq libraries were prepared as previously described (Henriques et al., 2013; Nechaev et al., 2010).

value: 3rd instar larvae

value: Whole larvae

tag: Sex

value: Male

value: HWT

tag: target molecule

value: Short nascent capped RNA


title: HWT Start-seq females NextSeq rep2

value: Female


title: nonactivated IJ rep 1


title: HWT Start-seq males NextSeq rep2


title: HWT Start-seq mixed NextSeq rep2

value: Mixed


title: HWT Start-seq females NextSeq rep1


title: HWT Start-seq mixed NextSeq rep1


title: K36R Start-seq males NextSeq rep2


title: 15h in vivo activated IJ rep 3

source_name: whole individual worm, infective juvenile, in vivo activated, 15h


title: 15h in vivo activated IJ rep 2


title: K36R Start-seq females NextSeq rep2


title: K36R Start-seq females NextSeq rep1


title: K36R Start-seq males NextSeq rep1


title: K36R Start-seq mixed NextSeq rep2


title: chemostat STR-PFR culture PFR P3 28h, rep2

city: Stuttgart

first: Joana

middle: Danica

last: Simen

address: Allmandring 31

department: Institute of Biochemical Engineering

country: Germany

institute: University of Stuttgart

data_processing:
The bcl2fastq Conversion Software v. 1.8.4 from Illumina (http://support.illumina.com/downloads.html) was used to translate cluster intensity values into fastq files.
Sequenced reads were trimmed for adaptor sequence using Cutadapt v. 1.8.3, then mapped to E. coli K12 substr. W3110 whole genome from NCBI (GenBank: AP009048.1) using the RNA-seq aligner STAR v.2.4.2a.
Raw counts were derived for each gene based on the respective annotations available from UCSC genome browser (http://genome.ucsc.edu) for the chosen reference sequence applying HTseq-count v. 0.6.1 in the intersection-nonempty mode.
HTseq-derived raw count data were used as input after a non-specific filtering step that removed residual rRNAs and tRNAs  and low coverage genes with fewer than 2 counts per million (16-20 reads) in more than 25% of the dataset.
Genome_build: ASM1024v1 (NCBI E. coli K12 subst. W3110 genome (GenBank: AP009048.1))
Supplementary_files_format_and_content: xlsx file format includes raw gene counts for each sample before and after filtering for low coverage genes

organism: Escherichia coli

source_name: chemostat STR-PFR culture

growth_protocol: E. coli K12 substrain W3110 was used in this study. Bioreactor cultivations were performed using a two-compartment bioreactor system consisting of an STR (3L) with a recycle loop (PFR)  in defined medium at pH7 and 37°C. Bioreactor cultivations were carried out with a minimal medium containing (per liter) 19.0 g glucose, 1.0 g NaH2PO4.· 2 H2O, 2.6 g K2HPO4, 3.8 g (NH4)2SO4, and a trace element solution (0.11 g Na3C6H5O7, 0.00835 g FeCl3· 6 H2O, 0.00009 g ZnSO4· 7 H2O, 0.00005 g MnSO4· H2O, 0.0008 g CuSO4· 5 H2O, 0.00009 g CoCl2· 6 H2O, 0.0044 g CaCl2· 2 H2O, 0.1 g MgSO4· 7 H2O). The STR-PFR was operated as a chemostat with ammonia as the growth-limiting substrate. First, a reference steady state (µ=0.2 h−1) was established in the STR and sampled three times during a 16 h period following establishment of the steady state. Then, the PFR was added and samples for RNA-sequencing were aquired.

extract_protocol:
The RNeasy mini kit, with on column RNase-Free DNase I treatment, (both Qiagen, Hilden, Germany) was used for total RNA isolation (RNA ≥ 200 bases), according to the manufacturer’s protocol. RNA quality was determined by a Lab-on-a-Chip-System Bioanalyzer 2100 (Agilent, Boeblingen, Germany).
First, ribosomal RNA was depleted from 1 μg of total RNA using the Ribo-ZeroTM Magnetic Kit (Bacteria) (Epicentre, Madison, WI, USA). Next, mRNA libraries were prepared using the TruSeq mRNA Library Prep Kit (Illumina, San Diego, CA, USA). mRNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Sampling by directly placing the culture sample into RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany), following short centrifugation the pellet was stored at −70°C.

tag: sample port

value: PFR P3

tag: time

tag: replicate


title: K36R Start-seq mixed NextSeq rep1

tag: genotype


title: chemostat STR-PFR culture PFR P1 28h, rep2

value: PFR P1


title: 15h in vivo activated IJ rep 1


title: chemostat STR-PFR culture PFR P5 330min, rep2

value: PFR P5


title: chemostat STR-PFR culture PFR P5 25h, rep2


title: chemostat STR-PFR culture PFR P5 26h, rep2


title: Day 30 Control 1

city: Freiburg

first: Ori

last: Staszewski

address: Breisacher Str. 64

department: Institute of Neuropathology

institute: University Medical Center Freiburg

description: 30d-1R_AGTCAA_L003_R1_001.fastq.gz corresponds to sample 30d-1L

data_processing:
Base calling was performed with RTA 1.18.61 using standard parameters.
Fastq files were generated with CASAVA 1.8.2 using standard parameters.
Fastq files were aligned to the mouse genome using the subjunc aligner from the Rsubread package (Version 1.20.1) with default parameters (-nthread 12). Alignment was done against an index build on the Gencode M6 primary assembly fasta file.
Gene counts were determined using featureCounts from the Rsubread package (Version 1.20.1). Features were obtained from the Gencode M6 primary assembly gtf. Parameters used were: annot.ext=<path to gencode M6 primary assembly GTF>, isGTFAnnotationFile=TRUE, GTF.featureType="exon", GTF.attrType="gene_name", useMetaFeatures=TRUE, nthreads=12, reportReads=TRUE
Raw counts were filtered removing the genes with the lowest 32% of counts summed over all samples. Following voom transformation, differential gene expression was analyzed using the bioconductor package limma with default parameters.
Genome_build: Gencode M6 (GRCm38.p4)
Supplementary_files_format_and_content: fC_counts.txt, tab delimited text file, matrix with raw counts for each sample; voom_matrix.txt: tab delimited text file, matrix with logarithmic expression values after prefiltering and voom transformation ($E from the voom output).

source_name: ventral pons, contralateral, left

growth_protocol: SPF mouse facility

extract_protocol:
Total RNA was column purified using the RNeasy Mini Kit (QIAGEN) from the ipsilateral and contralateral ventral pons (containing the facial nucleus, and parts of the gigantocellular reticular nucleus, intermediate reticular nucleus, parvicellular reticular nucleus, alpha, and spinal trigeminal nucleus, oral) of CX3CR1GFP/wt mice that underwent unilateral facial nerve axotomy at 8 weeks of age, at the corresponding time points post axotomy.
A sequencing library was constructed using the Illumina TruSeq Stranded mRNA kit (starting material 300 ng total RNA). Library quality was assessed with the KAPA qPCR System (Peqlab) prior to sequencing.

treatment_protocol: unilateral facial nerve axotomy at the right stylomastoid foramen

tag: transgenic line

value: brain

value: 12 weeks


title: Day 30 Control 2

description: 30d-2R_AGTTCC_L003_R1_001.fastq.gz correspondsto sample 30d-2L


title: Day 30 Control 3

description: 30d-3R_ATGTCA_L003_R1_001.fastq.gz corresponds to sample 30d-3L


title: Day 30 Lesion 1

description: 30d-1L_CCGTCC_L003_R1_001.fastq.gz corresponds to sample 30d-1R

source_name: ventral pons, ipsilateral neurodegeneration, right


title: Day 14 Control 2

value: 10 weeks


title: chemostat STR-PFR culture PFR P3 25min, rep2


title: chemostat STR-PFR culture PFR P3 120min, rep2


title: chemostat STR-PFR culture PFR P5 45min, rep2


title: chemostat STR-PFR culture PFR P5 120min, rep2


title: chemostat STR-PFR culture PFR P1 25min, rep2


title: chemostat STR-PFR culture PFR P1 120min, rep2


title: chemostat STR-PFR culture PFR P5 210min, rep2


title: Day 30 Lesion 2

description: 30d-2L_GTCCGC_L003_R1_001.fastq.gz corresponds to sample 30d-2R


title: Day 14 Control 1


title: chemostat STR-PFR culture PFR P5 25min, rep2


title: Day 14 Lesion 3


title: chemostat STR-PFR culture PFR P5 75min, rep2


title: chemostat STR-PFR culture PFR P5 28h, rep2


title: Day 30 Lesion 3

description: 30d-3L_GTGAAA_L003_R1_001.fastq.gz corresponds to sample 30d-3R


title: chemostat STR-PFR culture PFR P5 10min, rep2


title: Day 14 Lesion 2


title: Day 14 Lesion 1


title: chemostat STR culture STR reference 3 , rep2

source_name: chemostat STR culture

value: STR


title: chemostat STR-PFR culture PFR P5 5min, rep2


title: Day 7 Control 3

value: 9 weeks


title: chemostat STR culture STR reference 2 , rep2


title: Day 14 Control 3


title: chemostat STR culture STR reference 1 , rep2


title: Day 7 Control 2


title: chemostat STR-PFR culture STR 26h, rep2


title: Day 7 Lesion 2


title: Day 7 Lesion 3


title: chemostat STR-PFR culture STR 25h, rep2


title: chemostat STR-PFR culture STR 28h, rep2


title: chemostat STR-PFR culture STR 330min, rep2


title: chemostat STR-PFR culture STR 120min, rep2


title: HWT Start-seq males MiSeq rep1


title: Day 7 Lesion 1


title: HWT Start-seq females MiSeq rep2


title: Day 0 Control 1

value: 8 weeks


title: Day 0 Control 3


title: chemostat STR-PFR culture STR 45min, rep2


title: Day 0 Control 2


title: Sample 4_CD161- Tconv

city: London

first: Chantal

last: Duurland

address: 30 Guilford Street

department: Infection, Inflammation and Rheumatology

institute: UCL Great Ormond Street Institute of Child Health

data_processing:
Samples were run on HiSeq 2500 in rapid run mode 50PE.
Sequencing reads were mapped to human NCBI genome Build 37.2 using tophat v2.0.10 and read count data for each transcript was obtained using the python scripts provided as part of the Dexseq package.
FPKM values were created using the normalisation tools included in the DESeq package followed by subsequent analysis using GeneSpring 13.0 (Agilent Technologies).
Note: Sample 2 CD161+ Treg was excluded due to low cDNA yield
Genome_build: human NCBI genome build 37.2
Supplementary_files_format_and_content: tab-delimited text files with FPKM values for each sample

source_name: PBMC_CD161- Tconv

extract_protocol:
Cell populations were sorted from total PBMC and RNA was extracted using Picopure RNA isolation kit (Thermo Fisher Scientific)
cDNA library was prepared using TruSeq RNA sample preparation kit (Illumina)

tag: subject status

value: Healthy Adult Control

tag: cell type

value: Peripheral Blood Mononuclear Cells (PBMCs)

tag: cell subtype

value: CD161- Tconv


title: chemostat STR-PFR culture STR 75min, rep2


title: Sample 4_CD161+ Tconv

source_name: PBMC_CD161+ Tconv

value: CD161+ Tconv


title: chemostat STR-PFR culture STR 210min, rep2


title: chemostat STR-PFR culture STR 25min, rep2


title: K36R Start-seq males MiSeq rep2

value: 3rd instar larvae

value: Whole larvae

value: Male


title: Sample 4_CD161+ Treg

source_name: PBMC_CD161+ Treg

value: CD161+ Treg


title: Sample 4_CD161- Treg

source_name: PBMC_CD161- Treg

value: CD161- Treg


title: Day 7 Control 1


title: chemostat STR-PFR culture STR 5min, rep2


title: Sample 3_CD161- Tconv


title: K36R Start-seq females MiSeq rep2

value: Female

value: Short nascent capped RNA


title: HWT Start-seq males MiSeq rep2

value: HWT


title: K36R Start-seq females MiSeq rep1


title: chemostat STR-PFR culture PFR P1 28h, rep1

value: PFR P1


title: Sample 3_CD161+ Tconv


title: HWT Start-seq females MiSeq rep1


title: HWT Start-seq mixed MiSeq rep2

value: Mixed


title: chemostat STR-PFR culture PFR P5 28h, rep1

value: PFR P5


title: Sample 3_CD161- Treg


title: HWT Start-seq mixed MiSeq rep1


title: Sample 2_CD161- Tconv


title: chemostat STR-PFR culture PFR P3 28h, rep1

value: PFR P3


title: K36R Start-seq mixed MiSeq rep2


title: chemostat STR-PFR culture STR 10min, rep2

value: STR


title: Sample 3_CD161+ Treg

value: Healthy Adult Control


title: Sample 2_CD161+ Tconv

value: Peripheral Blood Mononuclear Cells (PBMCs)


title: chemostat STR-PFR culture PFR P3 120min, rep1


title: chemostat STR-PFR culture PFR P5 210min, rep1


title: chemostat STR-PFR culture PFR P1 120min, rep1


title: chemostat STR-PFR culture PFR P5 25h, rep1


title: K36R Start-seq mixed MiSeq rep1


title: chemostat STR-PFR culture PFR P5 120min, rep1


title: Bach2KO rep2

city: Turku

first: Paulina

last: Budzynska

address: Kiinamyllynkatu 13

country: Finland

institute: University of Turku

data_processing:
Illumina Bcl2fastq 1.8.4 software   used for base-calling
the reads were aligned against the chicken reference genome (galGal4 Ensembl assembly, downloaded from Illumina’s iGenomes site) using TopHat version 2.1.0
The reads were associated with known genes based on Ensembl annotations and the number of reads associated with each gene was counted using subread package (v. 1.5.0).
The counts were normalised using the TMM normalisation method of the edgeR R/Bioconductor package. For statistical testing the data were further transformed into RPKM values (reads per kilobase per million) using the voom approach in the limma package.
Genome_build: Galgal4
Supplementary_files_format_and_content: abundance measurements in Clone18 and BACH2KO

organism: Gallus gallus

source_name: DT40 B cell line

growth_protocol: Chicken DT40 B cells were maintained in RPMI 1640 (Sigma life technologies) supplemented with 10% fetal calf serum (Hyclone), 1% chicken serum (BioWest), 50 µM β-mercaptoethanol, 1x GlutaMaxTM-I (Gibco®) and 1x Penicillin Streptomycin (Gibco®) in a humidified atmosphere with 5% CO2 at +40 °C

extract_protocol:
Total RNA was extracted from 5x106 cells of 3 independent cultures of Clone 18 and Bach2KO cells with RNeasy Mini Kit (Qiagen) with RNase-free DNase (Qiagen) treatment according to manufacturer’s instructions
Libraries were prepared from 100ng of total RNAs using standard Illumina protocols


title: Bach2KO rep3


title: K36R Start-seq males MiSeq rep1


title: Sample 1_CD161+ Tconv


title: Bach2KO rep1


title: Sample 1_CD161- Treg


title: Clone 18 rep3

value: wild type


title: chemostat STR-PFR culture PFR P5 45min, rep1


title: Sample 1_CD161+ Treg


title: chemostat STR-PFR culture PFR P1 25min, rep1


title: chemostat STR-PFR culture PFR P3 25min, rep1


title: Clone 18 rep2


title: K36R ATAC-seq rep3

description:
K36R ATAC-seq
processed file: K36R.ATAC.accepted_hits.sorted.hiquality.RPKM.bw

data_processing:
Alignment to reference genome using Tophat2 (RNA-seq) or Bowtie2 (Start-seq, ATAC-seq)
Generation of count tables for RNA-seq using htseq-count
Differential expression analysis of RNA-seq using DESeq2 or Cufflinks
Assignment of nucleotide position for Start-seq peaks using custom scripts
Generation of BigWig files for ATAC-seq samples using Deeptools
Analysis of meta-TSS profiles for ATAC-seq samples using Deeptools
Genome_build: dm3

extract_protocol:
Nuclei were isolated from whole larvae and incubated with Tn5 transposase from Nextera DNA Library Prep Kit (Illumina) according to manufacturer's protocols and standard ATAC-seq protocol (Buenrostro et al. 2013)
Libraries were prepared according to Illumina Nextera DNA Library Prep Kit protocol

value: genomic DNA


title: K36R ATAC-seq rep2

value: 3rd instar larvae


title: chemostat STR-PFR culture PFR P5 10min, rep1


title: chemostat STR-PFR culture PFR P5 25min, rep1


title: chemostat STR-PFR culture PFR P5 5min, rep1


title: Sample 1_CD161- Tconv

value: CD161- Tconv


title: Sample 2_CD161- Treg


title: chemostat STR culture STR  reference 3, rep1


title: K36R ATAC-seq rep1

value: Whole larvae


title: HWT ATAC-seq rep1

description:
HWT ATAC-seq
processed file: HWT.ATAC.accepted_hits.sorted.hiquality.RPKM.bw


title: chemostat STR culture STR  reference 1, rep1


title: HWT ATAC-seq rep2


title: K36R Nuclear RNA-seq female repD

description:
HWT vs. K36R Nuclear RNA-seq
processed file: dm3.nuc.DESeq2.diff

extract_protocol:
Total RNA was extracted from isolated nuclei using TRIzol reagent (Thermo Fisher) using manufacturer's protocols.
Libraries were prepared according to Illumina Tru-seq Stranded Total RNA-seq library prep protocol

value: Total Nuclear RNA


title: HWT ATAC-seq rep3


title: chemostat STR culture STR  reference 2, rep1


title: chemostat STR-PFR culture STR 26h, rep1


title: chemostat STR-PFR culture STR 28h, rep1


title: K36R Nuclear RNA-seq female repC


title: K36R Nuclear RNA-seq female repA


title: chemostat STR-PFR culture STR 330min, rep1


title: K36R Nuclear RNA-seq female repB


title: chemostat STR-PFR culture STR 25h, rep1


title: K36R Nuclear RNA-seq male repD

value: Male


title: chemostat STR-PFR culture STR 210min, rep1


title: Clone 18 rep1


title: K36R Nuclear RNA-seq male repC


title: chemostat STR-PFR culture STR 120min, rep1


title: chemostat STR-PFR culture STR 75min, rep1


title: K36R Nuclear RNA-seq male repB


title: HWT Nuclear RNA-seq female repD

value: Female


title: chemostat STR-PFR culture STR 45min, rep1


title: HWT Nuclear RNA-seq female repA


title: HWT Nuclear RNA-seq female repB


title: HWT Nuclear RNA-seq female repC


title: chemostat STR-PFR culture STR 5min, rep1


title: HWT Nuclear RNA-seq male repC


title: K36R Nuclear RNA-seq male repA


title: chemostat STR-PFR culture STR 25min, rep1


title: HWT Nuclear RNA-seq male repD


title: HWT Nuclear RNA-seq male repA


title: K36R Poly-A RNA-seq rep2

description:
HWT vs. K36R Poly-A RNA-seq
processed file: dm3.polyA.DESeq2.diff

extract_protocol:
Total RNA was extracted using TRIzol reagent (Thermo Fisher) using manufacturer's protocols.
Libraries were prepared according to Illumina Tru-seq Stranded Poly-A RNA-seq library prep protocol

value: PolyA RNA


title: chemostat STR-PFR culture STR 10min, rep1


title: HWT Nuclear RNA-seq male repB

value: HWT


title: ps_ops

city: Cambridge

first: Clare

last: Baker

address: Anatomy Building, Downing Street

department: PDN

institute: University of Cambridge

description: Single end opercular data biological replicate 3

data_processing:
High-quality reads were filtered based on the score value given in fastq files (FastQC vers. 0.10.1)
Read lengths were trimmed and reads with primer/adaptor sequences removed using Trimmomatic-0.30)
Reads were de novo assembled using Velvet (ver. 1.2.10) and OASES (ver. 0.2.08).  Velvet and OASIS were run using different k-mer lengths k27-k77 in k10 increments along with other default parameters. Results of these assemblies were merged using Velvet and OASES k-mer of K27.
Reads were mapped back to the transcriptome using Bowtie2 (vers. 2-2.1.0).
SAM files were cleaned to remove PCR duplicates and long-stretch (>85%) poly-A sequences.
Transcript counts for each sample were obtained with HTseq-count (vers. 0.5.4p3). A locus-to-transcript mapping file was used to collapse related transcripts and obtain locus-level counts.
The BioConductor package DESeq was used for differential expression analysis. A p-value of <0.1 was considered significant after adjustment (multiple testing using Benjamini-Hochberg)
Enrichment Analysis: Gene ontology (GO) and protein domain annotations were extracted from relevant UniProt records. Enrichment analysis was performed using hypergeometric mean and Bonferonni multiple testing correction. Enriched genes were classified using GO terms according to molecular function using the web-based resource PANTHER (Mi et al., 2016 Nucl. Acids Res. (2016) doi: 10.1093/nar/gkv1194).
Supplementary_files_format_and_content: Modrell_Suppl_File_1.xlsx: File contains protein uniprot id; Gene symbol; Gene description; Locus id; log2Fold and Fold change from the differential expression analysis; Padj value (p-value adjusted for multiple testing with Benjamini-Hochberg); Enriched protein domains and Enriched gene ontology terms
Supplementary_files_format_and_content: countTable_gt2_v3.txt: Raw count data for each loci for each biological replicate
Supplementary_files_format_and_content: pfish_v3_chordates.blast2.txt: Top blast results for all transcripts against chordates. These are in compact format with no alignments. There can be multiple hits per transcript.
Supplementary_files_format_and_content: pfish_v3_drerio.blast2.txt: Top blast results for all transcripts against zebrafish. These are in compact format with no alignments. There can be multiple hits per transcript.
Supplementary_files_format_and_content: pfish_v3_chordates_func.txt: Extracted functional mapping of top blast hits against chordates for the highest scoring transcript in each locus (where the blast score is <= 10e-5). Loci with no matches will not be represented in this file. The file also contains extracted GO terms and PFAM terms, where present.
Supplementary_files_format_and_content: pfish_v3_zfish_func.txt: Extracted functional mapping of top blast hits against zebrafish for the highest scoring transcript in each locus (where the blast score is <= 10e-5). Loci with no matches will not be represented in this file. The file also contains extracted GO terms and PFAM terms, where present.

organism: Polyodon spathula

source_name: Opercular, lateral line organ-enriched

extract_protocol:
Opercular (lateral line organ-enriched) and fin (no lateral line organs) tissues were manually dissected and pooled from different sets of 6-7 specimens each, to form three biological replicates. RNA was extracted using Trizol reagent (Ambion), as per manufacturer's protocol. RNA concentration was assessed using a Nanodrop N1000 spectophotometer and integrity using an Agilent 2100 Bioanalyzer (Cambridge Genomic Services).  Only samples with a RNA integrity number (RIN) greater than 9 were used for next-generation sequencing.
Libraries were prepared following the standard Illumina RNA Library Prep Kit.

treatment_protocol: Stage 46 yolk-sac larvae were preserved in RNALater (Ambion, Thermo Fisher Scientific Inc., Waltham, MA) overnight at 4oC. Excess solution was removed and sample were stored at -80oC until processing.

value: Opercular

value: Stage 46 yolk-sac larvae


title: K36R Poly-A RNA-seq rep3

value: Mixed


title: psop2

description: Opercular paired end data biological  replicate 2


title: ps_fins

description: Single end fin data biological replicate 3

source_name: Fin, no lateral line organs

value: Fin


title: psop1

description: Opercular paired end data biological replicate 1


title: K36R Poly-A RNA-seq rep1


title: psfin2

description: Paired end fin data biological replicate 2


title: HWT Poly-A RNA-seq rep2


title: HWT Poly-A RNA-seq rep3


title: psfin1

description: Paired end fin data biological replicate 1


title: HWT Poly-A RNA-seq rep1


title: horse_piRNA_fraction

status: Public on Mar 26 2017

city: Mississippi State

middle: W

last: Vandewege

state: Mississippi

address: 32 Creelman St

institute: Mississippi State University

description: small RNA

data_processing:
Adapters were trimmed and reads quality filtered using the FastX toolkit
Idenical small RNA  reads were collapsed into one sequence and counted
Small RNA equences were mapped to the genome using Bowtie allowing zero mismatches
RNA-seq reads were mapped to TE consensus libraries using Bowtie and RSEM
Expression levels measured at transcripts per million (TPM) were estimated from RSEM
Genome_build: equCab2 (GCA_000002305.1)
Genome_build: canFam3.1 (GCF_000002285.3)
Genome_build: Ept.fus.01 (GCA_000308155.1)
Supplementary_files_format_and_content: abundance

organism: Equus caballus

source_name: Testes

extract_protocol:
Testes were snap frozen from dog, horse, and a bat (Eptesicus fuscus).  Total RNA was extracted from ground tissue using standard TRIzol protocols.
The small RNA fraction was isolated and prepped for Illumina sequencing (50 bp, single-end).

value: Testes

value: adult


title: dog_mRNA_fraction

description: mRNA

organism: Canis lupus familiaris


title: dog_piRNA_fraction

description: small RNA


title: A375 C2_vem_repl1

city: Pisa

first: Romina

last: D'Aurizio

state: PI

address: via Moruzzi 1

department: IIT

country: Italy

institute: CNR

data_processing:
Raw sequences were de-multiplexed using the Illumina pipeline CASAVA v1.8.
Cutadapt v.1.2.1 was used for trimming adapter sequences off the raw reads. Reads with N calls were discarded using FASTX_Toolkit v 0.0.13.1 and only reads with all base calls showing a Phred score higher than 30 were retained for further analysis.
miRExpress v2.1.3 was exploited to identify pre-miRNA sequences (miRBase v.20) allowing at least 95% of sequence identity and a length tolerance range of 4bp for mapping.
Expression values were normalized to reads per million (rpm).
Supplementary_files_format_and_content: Tab-delimited text file including normalized read count per sample.

source_name: A375 C2 vem-resistant clone_vemurafenib

growth_protocol: 10^06 cells of A375 parental cell line or A375 C2 vem-resistant clone were seeded in 10cm dishes.

extract_protocol:
RNA was extracted using miRNEasy MINI Kit (Qiagen), following the manufacturer’s instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: After 24h, A375 and A375 C2 cells were treated with DMSO or 2uM vemurafenib for 48h. At the end of the treatment, cells were harvested.

tag: cell line background

value: melanoma cell line

value: vem-resistant clone (C2) derived from A375 cells

tag: treated with

value: 2uM vemurafenib


title: bat_mRNA_fraction

description: mRNA

organism: Eptesicus fuscus

tag: tissue


title: A375_vem_repl1

source_name: A375 cells_vemurafenib

value: control, parental cells


title: A375 C2_DMSO_repl1

source_name: A375 C2 vem-resistant clone_DMSO

value: DMSO


title: A375 C2_DMSO_repl2


title: A375_DMSO_repl2

source_name: A375 cells_DMSO


title: A375_vem_repl2


title: A375 C2_vem_repl2


title: RNA-seq 37C LB rep1

status: Public on Mar 25 2017

city: Daejeon

first: Byung-Kwan

last: Cho

address: 291 Daehak-ro

country: South Korea

institute: KAIST

data_processing:
Basecalls performed using CASAVA version 1.4
All sequencing reads were mapped to E. coli MG1655 reference genome (NC_000913) using CLC Genomics Workbench5 with the length fraction of 0.9 and the similarity of 0.99.
The expression levels were based upon the RPKM metric obtained from strand-specific manner.
Supplementary_files_format_and_content: RPKM is generated by in-house script

organism: Escherichia coli str. K-12 substr. MG1655

growth_protocol: Glycerol stocks of E. coli K12 strain MG1655 were inoculated into LB media  grown at 37 ℃ with constant agitation overnight. Cultures were diluted 1:100 into fresh minimal medium and then cultured at 37 ℃ to mid-exponential phase (OD600 nm ~ 0.6).

extract_protocol:
From the cells cultured in LB media at 37 ℃, total RNA was extracted using RNAsnapTM method, followed by the ethanol precipitation. rRNA was removed using ribo-zeroTM magnetic kit for bacteria in accordance with manufacturer’s instruction (Epicentre). rRNA removal was confirmed using ExperionTM system. Subsequently, 4 µg of the purified RNA was fragmented to sizes of ~300 bp using RNA fragmentation reagent (Ambion, Grand Island, NY).
50 ng of the fragmented RNA was converted to sequencing library using TruSeq® Stranded mRNA Sample Prep Kit in accordance with manufacturer’s instruction (Illumina).

treatment_protocol: No treatment


title: RNA-seq 37C LB rep2

source_name: Escherichia coli str. K-12 substr. MG1655


title: HT1080_Low Density_10cells/mm^3_IL-6/IL-8 treated

city: San Francisco

first: Minsuk

last: Kwak

address: 2340 Sutter St

institute: University of California San Francisco

data_processing:
Sequencing was done using Illumina HiSeq2000 platform. Basecalls performed using CASAVA version 1.4
Raw sequenced reads were trimmed for the Illumina adapater sequences using the ‘clip’ tool in Galaxy, and masked for low-complexity or low-quality sequence.
A Bowtie index for read alignment was generated based on the Feb.2009 (GRCh37/hg19) UCSC knownGene human transcriptome using Bowtie2 v2.3.1.
Single-end reads were directly aligned to this index using Bowtie2 embedded in RSEM v1.2.25. Aligned reads were used as inputs to estimate transcript- and gene-expression levels.
Gene-expression estimation were performed using a protocol from Li et al., BMC Bioinformatics, 2011., with the following command options: rsem-calculate-expression –p 8 –bowtie2 –output-genome-bam. Gene expression values were estimated as Transcripts Per Million (TPM), Fragments Per Kilobase of transcript per Million mapped reads (FPKM), and read counts.
For each library, FASTQC, RSeQC, and in-house scripts were used to assess read depth, gene coverage, and mapping rates, to exclude low quality libraries from further analysis.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: tab-delimited text files include read counts and TPM values for each sample

source_name: Connective tissue epithelial cells

growth_protocol: Human fibrosarcoma HT1080 cells (ATCC) were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, and 0.005% (w/v) gentamicin. The cells were maintined at 3737°C, 5% CO2 during cell culture.

extract_protocol:
The matrices were exposed to cell lysis buffer (Trizol) and mechanically broken down using a syringe. Total RNA isolation was performed with RNA MiniPrep kit (Zymo Research). mRNA was selected from total RNA using phosphorylated oligo-dT, and single-strand cDNA was generated from mRNA with SuperScript Reverse Transcriptase III. Double-strandcDNA generation was performed. The End-It DNA End-Repair kit (which includes End-it buffer, 1mM dNTPs, 1mM ATP, total enzyme mix) and T4 DNA ligase were combined for the DNA end-blunting, 5'-end phosphorylation, and ligation. phi29 DNA polymerase-based whole DNA pool amplication was adopted for mRNA transcriptome amplifcation.
cDNA libraries were prepared for sequencing using the standard protocol suggested by Illumina Nextera XT DNA Library Prep Kit

treatment_protocol: HT1080 cells were embedded in 2mg/ml type I collagen gel. Cell suspensions containing desired cell numbers in 1:1(v/v) ratio of culture media and reconstitution buffer were mixed with soluble rat-tail collagen I to obtain a final collagen I concentration of 2mg/ml. 1M NaOH was added to and mixed well to bring the pH to 7. the cell suspension was added to a 24-well coverslip-bottom cell-culture dish and maintained in a humidified incubator at 37°C to allow collagen polymerization. Cells were embedded at varying cell densities from 10 cells/mm^3 to 150 cells/mm^3, and low-density cells (10cells/mm^3) and high-density cells (50cells/mm^3) were treated with either IL-6, IL-8, or conditioned medium of IL-6 and IL-8.

value: Connective tissue-derived fibrosarcoma cells

tag: cell line

tag: passages

tag: culture condition

value: collagen type I matrices, 10 cells/mm^3

tag: cytokine treatment

value: Addition of IL-6 and IL-8 in a ratio of 5:2


title: HT1080_Low Density_10cells/mm^3_IL-6 treated

value: Addition of recombinant IL-6


title: HT1080_Low Density_10cells/mm^3_IL-8 treated

value: Addition of recombinant IL-8


title: HT1080_Low Density_10cells/mm^3


title: EV Rep 2

city: Denton

first: Rajeev

middle: K.

last: Azad

state: Texas

address: 1155 Union Circle #305220

institute: University of North Texas

data_processing:
Used Tophat and Bowtie for read alignment onto human reference genome and Cufflinks for obtaining the FPKMs and assessment of significance of fold change
Genome_build: NCBI build 37.2
Supplementary_files_format_and_content: Excel file; FPKMs for empty vector control and mNT suppressed breast cancer cell lines MCF-7

source_name: breast cancer

growth_protocol: Cells were grown in culture in complete DMEM liquid medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin and 0.25 ug/ml amphotericin B.  Cells were maintained in petri dishes in an incubator at 37 degrees C and 5% CO2.

extract_protocol:
Cells were grown to 80% confluence, medium was discarded and cells were washed with 1x PBS.  Cells were collected directly from plates and RNA was extracted using the Qiagen RNeasy Mini Kit (Cat.# 74104) according to manufacturers instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: NAF-1(-) cells were generated through Lipofectamine transfection using a 29-mer pGFP-V-RS plasmid from OriGene Technologies, previously described in Sohn et al. (2013). EV (empty vector) was the scrambled sequence negative control. Cells were grown under puromycin selection and stable knockdown lines were obtained by FACS, selecting for GFP.

value: breast cancer

tag: genotype/variation

value: Control


title: EV Rep 1


title: RNA140717RH_69

city: Portland

first: Priscila

last: Darakjian

state: OR

address: 3181 SW Sam Jackson Park Road, L470

department: Behavioral Neuroscience

institute: Oregon Health and Science University

data_processing:
Base Calling was done through Illumina CASAVA software
FASTQC was run for data inspection
Alignment: Sequence data were then aligned using STAR (Spliced Transcripts Alignment to a Reference [Dobin et al. 2013]) allowing for a maximum of three mismatches per 100bp read. For all samples > 85% of the reads uniquely aligned.outFilterMismatchNmax=3,outFilterScoreMinOverLread=0.33,outFilterMismatchNoverLmax=0.03, outFilterMultimapNmax=1.
Reads Count: Using the Bedtools suite, reads were aligned to known genomic features to generate counts at the gene level.
Normalization: Gene expression data were imported into the R application environment; upper-quartile normalization was performed using the edgeR Bioconductor package (Robinson et al. 2010). The read density threshold for inclusion in the data analyses for genes was 30 or approximately 1 count per million reads.
Genome_build: Mus musculus (mm10)
Supplementary_files_format_and_content: RNA140717RH_mm10_exon_coverage_for_GEO.xlsx: Provides the phenotype related to each sample as well as the normalized (upper quartile) read counts for each gene for each sample
Supplementary_files_format_and_content: RNA140717RH_mm10_gene_coverage_for_GEO.xlsx: Provides the phenotype related to each sample as well as the normalized (upper quartile) read counts for each gene for each sample

source_name: Dissected Tissue (Brain) - Striatum

extract_protocol:
Frozen brains were slightly thawed and dissected by hand under RNAse-free conditions. Using the optic chiasm as the rostral marker, a 2 mm coronal slice of brain tissue was isolated. Beginning at the medial ventral aspect of the striatum and recognizing that the striatum has a partial cone shape, the dissection moved dorsal 1 mm, followed by a cut to the lateral boundary of the striatum, with a final cut following the lateral-ventral boundary. The isolated tissue was immediately placed into 1 ml of Trizol (Invitrogen, Carlsbad, CA).
Library formation (polyA+, stranded) and sequencing were all performed according to Illumina’s specifications at the OHSU Massively Parallel Sequencing Shared Resource. Libraries were multiplexed 6 per lane, yielding approximately 25 to 30 million total reads per sample.

treatment_protocol: Animals underwent the standard 4-day DID trial with three 2 hour exposures to a single bottle of 20% ethanol and a final day of 4 hour exposure. The DID trial always began 3 hours into the dark phase. BECs were obtained at the end of the 4th hour. A subgroup (N=20) of the animals were retested 3 weeks later. The remaining animals, 18 males and 17 females were sacrificed 3 weeks later and used for gene expression analyses. All animals were sacrificed between 10 AM and 2 PM.

tag: group

value: HSNpt (control)

tag: Sex

value: Male


title: horse_mRNA_fraction

description: mRNA

tag: developmental stage


title: RNA140717RH_67


title: EV Rep 3


title: mNT(-) Rep 3

value: mNT suppressed


title: mNT(-) Rep 2


title: bat_piRNA_fraction

description: small RNA

value: Testes


title: A375_DMSO_repl1


title: HT1080_High Density_50cells/mm^3

value: collagen type I matrices, 50 cells/mm^3


title: RNA140717RH_66


title: RNA140717RH_64


title: RNA140717RH_62


title: RNA140717RH_57


title: RNA140717RH_65


title: RNA140717RH_60


title: RNA140717RH_61


title: mNT(-) Rep 1


title: RNA140717RH_58


title: RNA140717RH_63


title: RNA140717RH_59


title: RNA140717RH_55


title: RNA140717RH_56


title: RNA140717RH_68


title: RNA140717RH_52

value: Female


title: RNA140717RH_53


title: RNA140717RH_51


title: RNA140717RH_48


title: RNA140717RH_50


title: RNA140717RH_49


title: RNA140717RH_47


title: RNA140717RH_45


title: RNA140717RH_46


title: RNA140717RH_43


title: RNA140717RH_42


title: RNA140717RH_44


title: RNA140717RH_41


title: RNA140717RH_54


title: RNA140717RH_40


title: RNA140717RH_37


title: RNA140717RH_38


title: RNA140717RH_39


title: RNA140717RH_36


title: RNA140717RH_32

value: HDID1 (selected line)


title: RNA140717RH_35


title: RNA140717RH_29


title: RNA140717RH_34


title: RNA140717RH_31


title: RNA140717RH_30


title: RNA140717RH_25


title: RNA140717RH_26


title: RNA140717RH_23


title: RNA140717RH_21


title: RNA140717RH_20


title: RNA140717RH_22


title: RNA140717RH_17


title: RNA140717RH_16


title: RNA140717RH_18


title: RNA140717RH_14


title: RNA140717RH_33


title: RNA140717RH_15


title: RNA140717RH_12


title: S8_nonNeuronal_Nuclei

city: New York

first: Panos

last: Roussos

state: NY

address: 1470 Madison Ave

department: Psychiatry

institute: Icahn School of Medicine at Mount Sinai

data_processing:
library strategy: ATAC-seq
Reads were mapped to the gender appropriate reference genome hg19 using STAR aligner version 2.5.0, by disabling  spliced alignments with (setting --alignIntronMax 1) and prohibiting soft-clipping of the reads (setting --alignEndsType EndToEnd).
For each sample, this produced a coordinate-sorted BAM file of mapped paired-end reads. We excluded reads that: (1) mapped to more than one locus; (2) were duplicated; and/or (3) mapped to the mitochondrial genome.
Overall quality control metrics were calculated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), SPP (Kharchenko et al., 2008), Phantompeakqualtools (https://code.google.com/p/phantompeakqualtools/) and ChIPQC (Carroll et al., 2014) for each sample, including: (1) total number of initial reads; (2) number of uniquely mapped reads; (3) the rates of duplicated reads; (4) the rates of reads mapping to the mitochondrial genome; (5) the PCR bottleneck coefficient (PBC), which is an approximate measure of library complexity estimated as (non-redundant, uniquely mapped reads)/(uniquely mapped reads); (6) normalized strand cross-correlation coefficient (NSC) and relative strand cross-correlation coefficient (RSC), which are metrics that use cross-correlation of stranded read density profiles to measure enrichment independently of peak calling and; (7) fraction of reads in peaks (FRiP), which is the fraction of reads that fall in detected peaks (see below for peak calling).
Genome_build: hg19
Supplementary_files_format_and_content: BigWig files were generated from BAM files using the genomeCoverageBed and bedGraphToBigWig functions

extract_protocol:
50mg of frozen powdered brain tissue was homogenized in cold lysis buffer (0.32M Sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM, EDTA, 10mM Tris-HCl, pH8, 1 mM DTT, 0.1% Triton X-100) and filtered through a 40µm cell strainer. The flow-through was underlaid with sucrose solution (1.8 M Sucrose, 3 mM Mg(Ace)2, 1 mM DTT, 10 mM Tris-HCl, pH8) and subjected to ultracentrifugation at 24,000 rpm for 1 hour at 4°C. Pellets were thoroughly resuspended in 500µl DPBS and incubated in BSA (final concentration 0.1%) and anti-NeuN antibody (1:1000, Alexa488 conjugated, Millipore) under rotation for 1 hour, at 4 °C. Prior to FANS sorting, DAPI (Thermoscientific) was added to a final concentration of 1µg/ml. DAPI positive neuronal (NeuN+) and non-neuronal (NeuN-) nuclei were sorted into tubes pre-coated with 5%BSA using a FACSAria flow cytometer (BD Biosciences) equipped with a 70μm nozzle.
Briefly, 50,000 sorted nuclei were centrifuged at 500 ×g
for 10 min, 4°C. Pellets were re-suspended in transposase reaction mix (25 μL 2x TD Buffer (Illumina Cat #FC-121-1030) 2.5 μL Tn5 Transposase (Illumina Cat #FC-121-1030) and 22.5 μL Nuclease Free H2O) on ice. Samples were incubated at 37°C for 30 min and then purified using the MinElute Reaction Cleanup kit (Qiagen Cat #28204), eluting in 10µl of buffer EB. Following purification, library fragments were amplified using the Nextera index kit (Illumina Cat #FC-121-1011), using the following cycling conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermocycling at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute for a total of 5 cycles. In order to prevent saturation due to over-amplification, a 5µl aliquot was then removed and subjected to qPCR for 20 cycles to calculate the optimal number of cycles needed for the remaining 45 μL reaction. The additional number of cycles was determined as follows: (1) Plot linear Rn vs. Cycle
(2) Calculate the # of cycle that is corresponded to 1⁄4 of maximum fluorescent intensity. In general, we found adding 4-6 cycles to this estimate yielded optimal ATACseq libraries, as determined by analysis on Bioanalyzer High Sensitivity DNA Chips (Agilent technologies Cat#5067-4626). Libraries were amplified for a total of 9–14 cycles. Following PCR, ATACseq libraries were resolved on 2% agarose gels supplemented with SYBR green. Fragments ranging in size from 100bp-1Kbp were excised and purified (Qiagen Minelute Gel Extraction Kit – Qiagen Cat#28604). Libraries were quantified by Qubit HS DNA kit (Life technologies) and by quantitiative PCR (KAPA Biosystems Cat#KK4873) prior to sequencing. Libraries were sequenced on Hi-Seq2500 (Illumina) obtaining 2x50 paired-end reads.

tag: gender

tag: age of death (yrs)

tag: postmortem interval (hours)

tag: brain weight (grams)

tag: tobacco use

value: No

tag: Cause of death

value: Cardio respiratory failure

tag: hypertension

value: Yes

tag: diabetes (insulin dependent)

tag: diabetes (non insulin dependent)

value: Frontopolar prefrontal cortex

value: Non-Neuronal


title: RNA140717RH_27


title: RNA140717RH_28


title: S7_nonNeuronal_Nuclei

tag: tissue

tag: cell type


title: S6_nonNeuronal_Nuclei

value: Atrial fibrillation


title: RNA140717RH_11


title: RNA140717RH_24


title: RNA140717RH_9


title: S5_nonNeuronal_Nuclei


title: S4_nonNeuronal_Nuclei

value: Myocardial infarction


title: RNA140717RH_13


title: RNA140717RH_8


title: RNA140717RH_19


title: S3_nonNeuronal_Nuclei

value: Lung cancer


title: S2_nonNeuronal_Nuclei


title: RNA140717RH_7


title: S1_nonNeuronal_Nuclei


title: S8_Neuronal_Nuclei

value: Neuronal


title: S6_Neuronal_Nuclei


title: RNA140717RH_2


title: RNA140717RH_6


title: RNA140717RH_4


title: S5_Neuronal_Nuclei


title: RNA140717RH_5


title: S7_Neuronal_Nuclei


title: RNA140717RH_3


title: RNA140717RH_10


title: S4_Neuronal_Nuclei


title: S1_Neuronal_Nuclei


title: S3_Neuronal_Nuclei


title: RNA140717RH_1


title: S2_Neuronal_Nuclei


title: WT

city: Hefei

first: Shi

last: Yin

address: Huangshan Road 443

institute: The University of Science and Technology of China

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

source_name: Oocyte

extract_protocol:
Oocytes were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain

value: oocyte

tag: age

value: 2 weeks old

tag: genotype

value: wild type


title: 4C CTCF-delA-1

status: Public on Mar 24 2017

city: Bethesda

first: Michaela

last: Willi

address: 9000 Rockville Pike

department: NIDDK

institute: NIH

data_processing:
library strategy: 4C-seq
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then trimmed the first 5'end 25bp
Trimmed reads were mapped to BamHI enzyme based reduced mm10 genome
Mapped reads were processed with 4Cker R package to generate bedgraph files
Supplementary_files_format_and_content: bedGraph files were generated by 4Cker R package

source_name: 4C CTCF-delA

growth_protocol: Mammary gland is harvested from lactating day1 female mouse

extract_protocol:
Frozen-stored mammary tissues collected at lactation day 1 (L1) were ground into powder. Chromatin was fixed with formaldehyde (final 1%) for 15 min at room temperature, and then was quenched with glycine (final 0.125 M). Pellets were lysed in lysis buffer (10 mM Tris HCI pH 8.0, 10 mM NaCl, 0.5% Nonidet P-40) containing Phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors, incubated on ice for 30 min, and dounced using pre-chilled glass homogenizer, followed by another 10 min incubation on ice. After removal of supernatant, nuclei pellets were re-suspended in restriction endonuclease buffer 2 (NEB; 50 mM NaCl,10 mM Tris-HCl,10 mM MgCl2,1 mM Dithiothreitol; New Englnad Biolabs). SDS was added to a final concentration of 0.2% and samples was incubated for 1 hr at 37°C. 2% Triton X-100 was added, followed incubation for 1 hr at 37°C. Samples were incubated with 400 units each of restriction enzymes BamHI (New Englnad Biolabs) and were incubated overnight at 37°C. To enzyme inactivation SDS was added (final 1%) and incubated for 30 min at 65°C, followed by SDS-sequestration with 1% Triton X-100 at 37°C for 1 hr, T4 ligase (New England Biolabs) was added to each sample and incubated for 1 hr at 16°C. Ligated samples were treated overnight with proteinase K (20 mg/mL, Invitrogen) at 65°C and 1 hr at 37°C with RNase A (10 mg/mL, Thermo Fisher Scientific), and DNA fragments were purified by phenol-chloroform method.
DNA fragments from 3C were digested with NlaIII (New England Biolabs) overnight at 37°C and ligated using T4 ligase overnight at 16°C. DNA was purified by phenol/chloroform method and then amplified with site-specific primers linked to the Illumina DNA adaptors. Libraries for next generation sequencing were prepared and sequenced with HiSeq 2500 (Illumina)

tag: genotype/variation

value: CTCF site A mutant

value: lactating day1(L1)

value: mammary gland