title: 20120724_RPF-Seq_5dpf

status: Public on May 23 2013

city: Cambridge

first: Guo-Liang

last: Chew

address: 16 Divinity Ave BL 1019

department: Dept of Molecular and Cellular Biology

institute: Harvard University

description: size-selected on PAGE gel, ~30nt

data_processing:
Adapters trimmed using custom script
Reads that mapped to zebrafish rRNAs (SILVA rRNA database: http://www.arb-silva.de/) by Bowtie2 -N 1 -L 20 _k 20) were discarded
Remaining reads mapped to previously assembled zebrafish developmental transcriptome (Pauli et al. 2012) on top of the Zv9/danRer7 assembly of the zebrafish genome by Tophat2; no indels, no novel junctions, -M -g 10
Only RPFs of length 27nt to 32nt used. P-site positions determined to be +12 for 27-28nt RPFs, +13 for 29-31nt RPFs, +13 for 29-31nt RPFs, +14 for 32nt RPFs.
Custom scripts used to generate bw files of genome-wide ribosome profiles corresponding to P-site occupancy, in conjunction with BedTools and UCSC bedgraphToBedBed
Genome_build: Zv9 (danRer7)
Supplementary_files_format_and_content: bigwig files corresponding to P-site occupancy by ribosomes

organism: Danio rerio

source_name: Whole embryos

growth_protocol: 400-600 embryos per stage from TL/AB WT strains were allowed to grow at 28.5°C and staged according to Kimmel et al. Dev. Dyn. 1995

extract_protocol:
Embryos were lysed by repeated micropipetting in 1.5ml of cold polysome buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1mM dithiothreitol, 100 μg/ml cycloheximide) with added 0.5% Triton X-100, 500 μg/ml GMP-PNP, 24 U/ml TurboDNase (Ambion AM2238), incubated with agitation for 10 min at 4°C, and clarified by centrifugation at 1300 rcf for 10 min at 4°C. 20μl RNAseI (Ambion AM2294) was added to the 1.5 ml of supernatant and incubated for 30 min at 37°C, then stopped by chilling on ice and addition of 40 μl of SuperaseIn (Ambion AM2694). Footprinted samples were pelleted through a sucrose cushion (1M sucrose in polysome buffer with added 100 U/ml SuperaseIn) by centrifugation at 260,000 rcf for 4.5 hours at 4°C, and resuspended in 800 μl 10mM Tris pH 7.4 with 1% SDS. RNA was purified by hot acid phenol/chloroform extraction and precipitated by standard ethanol precipitation.
Libraries were prepared essentially as described by Ingolia et al. Cell 2011

treatment_protocol: Embryos were quickly washed in ice cold PBS and flash frozen in liquid nitrogen

value: 5 days post fertilization

value: TL/AB

tag: target molecule

value: ribosome footprinted RNA


title: mule3703_day33_CG_RNAseq

status: Public on May 24 2013

type: SRA

city: Ithaca

first: Andrew

last: Clark

address: 227 Biotech Building

country: USA

institute: Cornell University

description:
Mule: Offspring of male donkey and female horse.
Illumina mRNA-seq

library_source: transcriptomic

data_processing: Alignment: The reads were truncated to 40 bp and aligned to the horse reference genome (UCSC version equCab2) using Burrows-Wheeler Alignment (BWA) with a maximum of four mismatches. On average, 67.1% of the reads were mapped to the horse reference genome.

molecule: total RNA

organism: Equus asinus x Equus caballus

source_name: mule embryonic day 33 chorionic girdle sample

extract_protocol: The mRNA-seq libraries were made with 2.5 ug of starting total RNA samples using the mRNA-Seq 8-Sample Prep Kit (Illumina Inc., CA), following the Illumina protocol for mRNA sequencing sample preparation.

tag: tissue

value: chorionic girdle

tag: developmental stage

value: embryonic day 33


title: 20120724_RPF-Seq_Bud

state: MA

description: size-selected on PAGE gel, ~30nt

value: Bud

tag: strain


title: NPC_Rep2_5C_library

city: Worcester

first: Jennifer

middle: Elizabeth

last: Phillips-Cremins

address: 364 Plantation Street

department: Program in Systems Biology and Department of Biology

institute: University of Massachusetts Medical School and Emory University

description: 3C and 5C

library_source: genomic

data_processing:
Reads were aligned to a pseudo-genome consisting of all 5C primers using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) (Langmead, 2010).  To account for poor quality reads, sequences were required to have only one unique alignment and 5 and 3 bases were trimmed from the 5’ and 3’ ends of the read, respectively.  Interactions were counted when both paired end reads could be uniquely mapped to the 5C primer pseudo-genome. Only interactions between forward-reverse primer pairs were tallied as a true count, as forward-forward or reverse-reverse primer pairs represent an artifact in the 5C procedure.
To account for bias intrinsic to all 3C-based methods, as well as to 5C specifically, we developed a probabilistic model that simultaneously captures the non-biological contribution of specific primers and the distance-dependent background level of non-specific chromatin interactions (Imakaev et al., 2012; Yaffe and Tanay, 2011).  For each region, model parameters describing the expected value and signal-dependent variance of each interaction under a normal-lognormal distribution were learned using stochastic gradient descent. Using the learned parameters, empirical p-values were computed under this distribution via Monte Carlo simulation for each observed interaction count.  Interaction scores were derived using the inverse cumulative density function for a standard normal distribution.  The resulting p-values and derived interactions scores are directly comparable within and between experiments and allow for robust detection of fragment-to-fragment looping interactions that are significant above the expected background signal.
The processed data file contains the following information for each forward-reverse primer combination possible in cis:  “Region” (5C Region), “Fprimer” (Forward primer ID), “Rprimer” (Reverse primer ID), “Distance” (mid-to-mid distance between fragments represented by forward and reverse primers), “Raw” (raw number of reads for a particular forward-reverse primer combination), “_.Predicted” (expected number of reads modeled from learned parameters for a particular forward-reverse primer combination), “_.PE” (primer-corrected counts for a particular forward-reverse primer combination), “_.DD”  (primer-corrected and distance-corrected counts for a particular forward-reverse primer combination), “_.Pvalue” (pvalue assigning significance to the counts for a particular forward-reverse primer combination after comparison to the expected distribution of counts at that distance scale), and “_.InteractionScore” (for each forward-reverse primer combination, interaction scores were derived from pvalues using the inverse cumulative density function for a standard normal distribution).  Processed data file includes information for 2 replicates of ES cells and 2 replicates of ES-derived NPCs (ES_Rep1, ES_Rep2, NPC_Rep1, and NPC_Rep2).

molecule: genomic DNA

organism: Mus musculus

source_name: Neural progenitor cells

growth_protocol: Murine V6.5 ES cells were expanded on Mitomycin-C-inactivated MEF feeder layers in pluripotent media supplemented with LIF.  After initial expansion, ES cells were passaged 2-3 times on 0.1% gelatin to remove contaminating feeder cells.

extract_protocol: 3C templates were generated using HindIII as previously described (Dekker et al., 2002; van Berkum and Dekker, 2009). 5C libraries were generated from 3C templates using an alternating primer design across 1-2 Mb regions around Oct4, Nanog, Sox2, Nestin, Olig1-Olig2, Klf4, and a gene desert negative control  (described previously Dostie and Dekker, 2007; Dostie et al., 2006; van Berkum and Dekker, 2009).

treatment_protocol: V6.5 ES cells were differentiated into neural progenitor cells (NPCs) using established techniques (Meissner et al., 2008; Mikkelsen et al., 2007; Okabe et al., 1996).  Briefly, after expansion, ES cells were trypsinized and cultured in bacterial dishes for 4 days in the absence of LIF to promote embryoid body formation.  Embryoid bodies were then plated on tissue-culture plastic dishes and cultured in serum-free, defined media (ITSFn).  After 5-7 days selection in ITSFn, adherent cells were trypsinized, triturated to a single cell suspension, and re-plated on tissue culture dishes coated with 15 ug/ml poly-L-ornithine (Sigma) and 1 ug/ml human plasma fibronectin (Gibco).  NPCs were further propagated for 2-4 days in DMEM/F12 supplemented with 25 ug/ml insulin, 50 ug/ml human APO transferrin, 30 nM sodium selenite, 20 nM progesterone (Sigma), 100 nM putrescine (Sigma), 1 ug/ml laminin (Sigma), 10 ng/ml Fgf2 (R&D Systems), and 1x penicillin/streptomycin. NPCs were ~60-75% confluent at the time of fixation with 1% formaldehyde before downstream assay.

tag: cell type

value: ES-derived neural progenitor cells

value: 129SvJae x C57BL/6

tag: gender

value: male

tag: chip antibody


title: 20120724_RPF-Seq_256Cell

description: size-selected on PAGE gel, ~30nt

value: 256 cells


title: 20120724_RPF-Seq_1KCell

description: size-selected on PAGE gel, ~30nt

value: 1k cells


title: 20120724_RPF-Seq_Dome

description: size-selected on PAGE gel, ~30nt

value: Dome


title: 20120724_RPF-Seq_2-4Cell

description: size-selected on PAGE gel, ~30nt

value: 2-4 cells


title: NPC_input DNA

description: ChIP-seq

data_processing: Sequences were aligned to NCBI Build 37 (UCSC mm9) using default parameters (-v1 -m1) in Bowtie.  Only sequences that mapped uniquely to the genome were used for further analysis.  Model-based Analysis for ChIP-Sequencing (MACS) was used for peak calling (http://liulab.dfci.harvard.edu/MACS/00README.html) (Zhang et al., 2008). Default parameters were used with a p-value cutoff of P<1E-8. A background whole cell extract input control was used to assess significance.

extract_protocol: Chromatin immunoprecipitation (ChIP) was performed as previously described with minor modifications (Kagey et al., 2010).  Briefly, 50x106 NPCs were lysed and chromatin was sonicated with a Branson Sonifier 250 for 30 x 12 sec pulses. Chromatin was pre-cleared with Protein A Sepharose beads and then immunoprecipitated with approximately 10 μg of the appropriate antibody coupled to Protein A sepharose beads.  For Smc1a ChIP using Bethyl Laboratories (A300-055A) affinity purified rabbit polyclonal antibody, beads were washed 1X with LB3, 1X with 20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 0.1% SDS, 1%Triton X-100, 1X with 10mM Tris-HCl pH8, 250nM LiCl, 2mM EDTA, 1% NP40 and 1X with TE containing 50 mM NaCl.  For CTCF ChIP using an Upstate 07-729 rabbit polyclonal antibody, beads were washed 7 times with RIPA buffer and 1X with TE containing 50 mM NaCl.  Bound complexes were eluted from the beads by heating at 65°C for 15 min with occasional vortexing.  Cross-links were reversed by overnight incubation at 65°C. Whole cell extract DNA reserved from the sonication step was also treated for crosslink reversal. ChIP-seq libraries were prepared for sequencing as recommended by Illumina/Solexa.


title: CEC Culture

city: Singapore

first: Zhenzhi

last: Chng

address: 8A Biomedical Grove #06-06

country: Singapore

institute: Institute of Medical Biology

data_processing:
Reads were alighned with ABI Bioscope version 1.3. Reads were first filtered for rRNA and tRNA samples and then aligned to the human genome (hg19.)
The uniquely aligned read counts (wt.merge.score.zone=4) were used for the calculation of RPKM and RPM values based on RefSeq annotation.
Genome_build: hg19
Supplementary_files_format_and_content: RPKM values in text format.

organism: Homo sapiens

source_name: corneal endothelial cell culture

growth_protocol: Research corneas were preserved and transported in Optisol-GS at 4°C, and were used within 13 days from preservation. Culture of Human Corneal Endothelial Cells: CEC-DM layer was digested enzymatically in collagenase A (2 mg/ml) for at least 2 hours and up to 6 hours. This allowed full detachment of the CECs from the DM, which tended to conglomerate into tightly-packed CEC clusters. The CEC clusters were rinsed once in PBS and further dissociated in TrypLE Express (TE) for 5 minutes. Cell pellets collected after a mild centrifugation (800 g for 5 minutes) were plated in culture-ware coated with FNC coating mixture. Isolated cells were left to adhere overnight in a stabilization medium made up of Human Endothelial-SFM supplemented with 5% FBS and 1x anti-biotic/anti-mycotic. Adhered hCECs were then cultured in F99 medium containing Ham’s F12 and M199, mixed in a 1:1 ratio, supplemented with 5% FBS, 20 μg/ml ascorbic acid, 1x Insulin-Transferrin-Selenium, 1x anti-biotic/anti-mycotic and 10 ng/ml bFGF. When the cultured cells reached 80-90% confluence, they were re-exposed to the stabilization medium for at least one week before passaging. The inclusion of this final step enhanced the general morphology of cultured hCECs (unpublished observation; manuscript in preparation). Confluent hCECs were passaged using TE, and seeded onto FNC-coated culture ware at a plating density of approximately 10,000 cells per cm2 for subsequent passage. All incubation and cultivation of hCECs were carried out in a humidified incubator at 37°C containing 5% CO2 unless otherwise stated. Fresh media were replenished every two days.

extract_protocol:
For direct RNA extraction of donor tissues, isolated CEC-DM and corresponding corneal stroma button, punched out from the donor cornea tissue using an 8.0-mm diameter trephine, were rinsed once in PBS and placed directly into 1ml Trizol reagent. It should be noted that although most of the corneal epithelium spontaneously sloughed off the cornea surface during the transport and process of the cornea, remnant cells of the basal corneal epithelium might still be present on the corneal stroma button used for RNA extraction. Tissues were homogenized using a hand-held homogenizer before addition of 200ul of chloroform. After vigorous shaking, samples were spun at 13000 rpm for 15min at 4°C. The upper phase, containing total RNA, was transferred to a new tube containing an equal volume of 70% ethanol. The resulting solution was transferred into a QIAGEN RNeasy column and procedures were performed as per manufacturer’s protocol with a DNAse digestion step incorporated.
Libraries were prepared using an AB Demonstrated Protocol similar to the one reported in Tang et al 2009.  Starting amount of RNA was 100pg. SOLiD library preparation kit was used for library generation.

treatment_protocol: Research corneas were incubated in three washes of antibiotic/antimycotic solution in PBS, 15 minutes each. Corneoscleral rims were placed endothelial-side-up on a disposable cornea vacuum punch (Ripon, England), and mildly stabilized by the vacuum suction created. A brief 30 seconds treatment with Trypan Blue solution (0.1%) was used to delineate Schwalbe’s line. The CEC-DM layer was carefully stripped off, approximately 1 mm anterior to the Schwalbe’s line (away from the trabecular meshwork) from the posterior stroma under the dissecting microscope (Nikon, Japan).

value: corneal endothelial cells culture


title: CTCF_NPC_ChIPSeq

description: ChIP-seq

value: CTCF (Millipore;  07-729 DAM1772428)

tag: catalog/lot#


title: Corneal Stroma

source_name: Pool of corneal stroma from 5 young donors

value: corneal stroma


title: CEC Young

source_name: Pool of corneal endothelial cells from 5 young donors

value: corneal endothelial cells


title: CEC Old

source_name: Pool of corneal endothelial cells from 5 old donors


title: dme_TollM2_input_2to4h_1

city: Kansas City

first: Julia

last: Zeitlinger

state: MO

institute: Stowers Institute for Medical Research

description: input DNA from Toll10b embryos with one copy of plasmid transgene (pnrenhancer-tupPr-yellow, named M2)

data_processing:
Reads were aligned to the UCSC dm3 genome with the addition of specific BAC sequences as pseudo-chromosomes with bowtie v0.12.7. Only uniquely aligning reads with up to two mismatches were retained. For paired-end samples, successfully aligned mate pairs were joined to a single fragment.
Genome Build:
toll10b_input.bed: dm3

organism: Drosophila melanogaster

source_name: input DNA from Toll10b embryos expressing the pnrenhancer-tupPr-yellow plasmid

growth_protocol: D. melanogaster embryos were collected and aged on yeasted apple juice plates at 25 °C and 60 % humidity.

extract_protocol: ChIP-seq: DNA-protein-complexes were isolated with specific antibodies. Sequencing libraries were prepared following Illumina's instructions: DNA was end repaired using T4 DNA polymerase, DNA polymerase I large fragment (Klenow polymerase) and T4 polynucleotide kinase. A single 3'-A overhang was added to the blunted ends using Klenow 3'-5' exo- polymerase. Adapters with single 3'-T overhangs were ligated to the adenylated fragments, and after a size-selection step on a 2 % agarose gel (150-250 bp) the adapter modified DNA was PCR-amplified. Following cluster generation on the flowcell surface, the sequencing libraries were sequenced following Illumina's protocol.

treatment_protocol: no treatment, only fixation of embryos in formaldehyde

value: whole Toll10b embryos

tag: tissue type

value: mesodermal

tag: genotype

value: maternally Toll10b/+, zygotically expressing the pnrenhancer-tupPr-yellow plasmid

value: none (input)


title: dme_gd144_pol_2to4h_1

description: Pol II IP DNA from gd7 embryos with one copy of a 25kb TwistBAC where the twi promoter has been replaced by the snail core promoter (Bac144) and the twi coding region replaced by the yellow sequence (Bac144)

data_processing:
Reads were aligned to the UCSC dm3 genome with the addition of specific BAC sequences as pseudo-chromosomes with bowtie v0.12.7. Only uniquely aligning reads with up to two mismatches were retained. For paired-end samples, successfully aligned mate pairs were joined to a single fragment.
Genome Build:
gd144_pol.bed: dm3

source_name: Pol II IP DNA from gd7 embryos expressing the transgene twiBAC-snaPr-Yellow

value: whole gd7 embryos

value: dorsal ectoderm

value: maternally gd7/gd7 mutants and zygotically expressing the transgene twiBAC-snaPr-Yellow

value: Pol II (4H8, Millipore)


title: dme_TollM2_Pol_2to4h_1

description: Pol II IP DNA from Toll10b embryos with one copy of plasmid transgene (pnrenhancer-tupPr-yellow, named M2)

data_processing:
Reads were aligned to the UCSC dm3 genome with the addition of specific BAC sequences as pseudo-chromosomes with bowtie v0.12.7. Only uniquely aligning reads with up to two mismatches were retained. For paired-end samples, successfully aligned mate pairs were joined to a single fragment.
Genome Build:
toll10b_pol.bed: dm3

source_name: Pol II IP DNA from Toll10b embryos expressing the pnrenhancer-tupPr-yellow plasmid


title: dme_gd_pol_2to4h_1

description: Pol II IP DNA from gd7 embryos with one copy of a 25kb TwistBAC  where the twi coding region has been replaced by the yellow sequence

data_processing:
Reads were aligned to the UCSC dm3 genome with the addition of specific BAC sequences as pseudo-chromosomes with bowtie v0.12.7. Only uniquely aligning reads with up to two mismatches were retained. For paired-end samples, successfully aligned mate pairs were joined to a single fragment.
Genome Build:
gd_pol.bed: dm3

source_name: Pol II IP DNA from gd7 embryos expressing the transgene twiBAC-Yellow

value: maternally gd7/gd7 mutants and zygotically expressing the transgene twiBAC-Yellow


title: dme_gd_input_2to4h_1

description: input DNA from gd7 embryos with one copy of a 25kb TwistBAC where the twi coding region has been replaced by the yellow sequence

data_processing:
Reads were aligned to the UCSC dm3 genome with the addition of specific BAC sequences as pseudo-chromosomes with bowtie v0.12.7. Only uniquely aligning reads with up to two mismatches were retained. For paired-end samples, successfully aligned mate pairs were joined to a single fragment.
Genome Build:
gd_input.bed: dm3

source_name: input DNA from gd7 embryos expressing the transgene twiBAC-Yellow


title: 20120724_RPF-Seq_Shield

description: size-selected on PAGE gel, ~30nt

value: Shield

value: TL/AB

value: ribosome footprinted RNA


title: ctrl_WT-wRPB2_tet

status: Public on May 22 2013

city: New York

first: Harm

middle: van

last: Bakel

state: New York

address: One Gustave L. Levy Place, Box 1498

department: Genetics and Genomic Sciences

institute: Mount Sinai School of Medicine

description: Monococcal nuclease treated chromatin profile for wild-type strain

data_processing:
library strategy: Nuc-seq
Illumina Casava1.7 software was used for basecalling.
Sequenced reads were trimmed for adaptor sequence using cutadapt and low-quality sequences were trimmed using custom perl scripts. Reads were then mapped to the May 2008 SGD S. cerevisiae genome assembly using bowtie v0.12.7 with parameters --best --strata -e 140 -a -m 1 -n 2 -5 4 -3 10 –maxins 1000
The midpoint of each mapped read pair from a mononucleosomal fragment was used as an estimate of the nucleosome center position.
Genome_build: Saccharomyces Genome Database (SGD), may 2008 build
Supplementary_files_format_and_content: Processed data files are in wig format and contain the mapped nucleosome midpoint positions. Scores represent the number of mapped midpoints.

organism: Saccharomyces cerevisiae

source_name: Wild-type control for RPB2_tet strain

growth_protocol: For controls of Tet promoter-shutoff strains, doxycyline was added at 10μg/ml for 24 h in order to obtain down-regulation for the gene of interest. Cells were then diluted to an OD600 of 0.2 and grown in the presence of doxycycline (10μg/ml) until mid-log phase.

extract_protocol:
Cells were crosslinked by direct addition of methanol-free formaldehyde (Polysciences) to a final concentration of 2% for 30 min while shaking at 30 °C. The reaction was quenched by adding glycine to a final concentration of 125 mM for 5 min. Cells were pelleted, washed with 20 mL phosphate buffered saline solution once, and resuspended in 6 mL of [1 M sorbitol, 50 mM Tris 7.4] with freshly added 10 mM β-mercaptoethanol in a 15 mL conical tube. Zymolyase (20T, TakaRa Biotechnology Co., Ltd, Japan) was added to a final concentration of 0.25 mg/mL and cells were spheroplasted at 30 °C while gently rolling for 30 min. After zymolyase treatment, cells were pelleted and resuspended in 4 mL of [1 M sorbitol, 50 mM NaCl, 10 mM Tris 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40] with freshly added 1 mM β-mercaptoethanol and 500 μM spermidine. Spheroplasts were divided into 6 aliquots of 300 μL each and transferred into 1.5 mL Eppendorf tubes. Micrococcal nuclease (MNase; Worthington) dissolved in water at 0.1 U/μL stock was added to the tubes at concentrations of 0, 25, 50, 75, 100, and 150 U per sample. The digestion reactions were incubated at 37 °C for 45 min and reactions were stopped by adding 75 μL of [5% SDS, 50 mM EDTA]. Remaining proteins were digested by adding 3 uL of 20 mg/mL proteinase K solution (Qiagen) was added to each tube for an overnight incubation at 65 °C. DNA from each MNase aliquot was isolated by phenol/chloroform extraction, concentrated via ethanol precipitation and treated with RNaseA (Fermentas) at a final concentration of 1mg/ml for 1 hour. Samples were then separated on a 2% agarose gel and bands corresponding to ~147bp (mono-nucleosomal fragments) were gel-extracted using the QIAquick kit (Qiagen) for each of the digestions.
Mononucleosome fragments were end-repaired, followed by amplification-free adaptor ligation and size selection on a 2% agarose gel. Clusters were generated on a single-read flowcell using Illumina’s cBot, and sequenced as 2x36 nt paired-end reads using an Illumina Genome Analyzer IIx instrument.

value: BY4741, MATa his3-del1 leu2-del0 met15-del0 URA3::CMV-tTA

tag: genotype/variation

value: Wild-type control for RPB2_tet strain


title: No-TAP

city: Rehovot

first: Gal

last: Haimovich

address: Belfer bldg. 416

department: Dept. Molecular Genetics

country: Israel

institute: Weizmann Institute of Science

description: yeast cells grown in YPD

data_processing:
Peconic Bioinf Guide (http://www.peconicgenomics.com/CHIP-exo%20services.html)
tab files were converted to wig file using a custom script
Genome_build: saccharomyces_cerevisiae_R56-1-1_20070407 (http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/)

source_name: yeast cells grown in YPD

growth_protocol: cells grown in YPD

extract_protocol:
1% formahldehyde-fixed cells were lysed in buffer 50 mM HEPES 150 mM NaCl, 2 mM EDTA (pH 8.0), 1% Triton X‐100  0.1% NaDeoxycholate. Lysis done with zirconium silicate beads for 6min in mini-beadbeater
Shear the chromatin to a median size of 250‐300 bp by sonication: using Diagenode  Bioruptor Standard (power setting: high, 30 seconds on / 30 seconds off, 30 cycles)
ChIP was performed with Rabbit IgG sepharose beads (Bethyl P120-101). After several rounds of wash, beads were treated with enzymes in the following order: T4 DNA polymerase, then T4 Polynucleotide Kinase, then Klenow Fragment (3’→5’ exo‐) (these steps are for polishing the DNA ends), then ligation to PCR adaptors and elongation using phi29 DNA polymerase
DNA on beads was then digested with Lambda exonulease, followed by RecJf Exonuclease. DNA was eluted from beads at 65C.
For complete protocol see: Rhee & Pugh (2012) curr. Protoc. Mol. Biol. Chapter 21:Unit 21.24. doi: 10.1002/0471142727.mb2124s100
To complete library construction for high‐throughput sequencing, the DNA is primer‐extended and ligated with a second sequencing adaptor. After gel‐purification and PCR, the amplified library is ready for high‐throughput sequencing
For complete protocol see: Rhee & Pugh (2012) curr. Protoc. Mol. Biol. Chapter 21:Unit 21.24. doi: 10.1002/0471142727.mb2124s100

tag: background strain

value: no TAP tag


title: Dcp2-TAP

description: yeast cells grown in YPD


title: Lsm1-TAP

description: yeast cells grown in YPD


title: ctrl_WT_del_rep03

source_name: Wild-type control for deletion strains

growth_protocol: For deletion strain controls, 5 mL cultures were grown overnight at 30 °C in YPD. Cells were diluted to OD600 0.2/mL in 400 mL of YPD media the next morning, and grown to mid-log phase (OD600 0.8-1.0/ml) in 1L flasks at 30 °C while shaking, at which point RNA and nucleosomal DNA were isolated.

value: BY4741, MATa his3-del1 leu2-del0 met15-del0 ura3-del0

value: Wild-type control for deletion strains


title: ctrl_WT_del_rep02


title: TBF1_ts

description: Monococcal nuclease treated chromatin profile for TBF1 temperature-sensive strain grown at restrictive temperature

growth_protocol: Temperature-sensitive strains were grown at 22°C (permissive temperature) until mid-log phase. An equal volume of hot medium was then added to equilibrate the culture to 37°C (restrictive temperature), followed by a further 3 to 7 hours incubation at this temperature until a difference in OD between the mutant strain and its corresponding wild-type control became apparent.

value: BY4741, MATa his3-del1 leu2-del0 met15-del0 ura3-del0 KanMX::tbf1-URA3


title: SET2_del_rep1

description: Monococcal nuclease treated chromatin profile for SET2 deletion strain

source_name: SET2 deletion strain

growth_protocol: For deletion strains, 5 mL cultures were grown overnight at 30 °C in YPD. Cells were diluted to OD600 0.2/mL in 400 mL of YPD media the next morning, and grown to mid-log phase (OD600 0.8-1.0/ml) in 1L flasks at 30 °C while shaking, at which point RNA and nucleosomal DNA were isolated.

value: SET2 deletion strain


title: SWR1_del

description: Monococcal nuclease treated chromatin profile for SWR1 deletion strain

source_name: SWR1 deletion strain

value: SWR1 deletion strain


title: SPT16_ts

description: Monococcal nuclease treated chromatin profile for SPT16 temperature-sensive strain grown at restrictive temperature

value: FY85, MATa his4-912delta lys2-128delta leu2-del1 ura3-52 spt16-197


title: RLF2_del_rep1

description: Monococcal nuclease treated chromatin profile for RLF2 deletion strain

source_name: RLF2 deletion strain

value: RLF2 deletion strain


title: ctrl_WT-wSPT16_ts

source_name: Wild-type control for SPT16_ts strain

growth_protocol: Controls for temperature-sensitive strains were grown at 22°C (permissive temperature) until mid-log phase. An equal volume of hot medium was then added to equilibrate the culture to 37°C (restrictive temperature), followed by a further 3 to 7 hours incubation at this temperature until a difference in OD between the mutant strain and its corresponding wild-type control became apparent.

value: FY85, MATa his4-912delta lys2-128delta leu2-del1 ura3-52

value: Wild-type control for SPT16_ts strain


title: CYC8_del_rep1

description: Monococcal nuclease treated chromatin profile for CYC8 deletion strain

source_name: CYC8 deletion strain

value: CYC8 deletion strain


title: ChIPSeq_H3K27me3_e11.5cortex

city: New Haven

first: Laura

last: DeMare

state: CT

address: 333 Cedar St

department: Genetics

institute: Yale University School of Medicine

data_processing:
ChIP-seq reads (E10.5 limb CTCF) were aligned to mm9 genome assembly using Bowtie (0.12.3) and only uniquely aligning reads were retained (bowtie -B 1 -p 8 --solexa1.3-quals). To call peaks, MACS (1.3.7) was supplied with both ChIP and input samples (input from GSE 30641) and run with the following parameters: --format=BOWTIE --tsize=74 --gsize=1860000000 --bw=150 --pvalue=0.00001 --mfold=10.
ChIP-seq reads (E11.5 limb CTCF, Smc1a, H3K27ac, H3K4m2, H327me3) were aligned to mm9 genome assembly using Bowtie (0.12.7) and only uniquely aligning reads were retained. For single-end sequencing data, the following parameters were used: bowtie -B 1 -p 8 -m 1.  For paired-end sequencing reads, an additional parameter was used (-X 1000). To call peaks for Smc1a and CTCF, MACS (1.4.1) was supplied with both ChIP and input samples and run with the following parameters: macs14 --pvalue=0.00001 --bw=300 -f BOWTIE -g mm. For CTCF, an additional parameter (mfold=5,30) was used.  For calling peaks with paired-end aligned reads, only the first mate pair was used for both the ChIP and input samples.  To call peaks for H3K27ac, H3K4me2, and H3K27me3, a custom perl script was implemented based on methodology from Mikkelsen et al. 2010 (PMID 20887899).
RNA-seq reads were aligned to the mm9 genome assembly using TopHat (v1.4.1) and supplying a known transcriptome index from UCSC (tophat -p 8 -g 1 --transcriptome-index=/home/GENOMES/transcriptome/mm9_known).
ChIA-PET reads were trimmed of ChIA-PET ligation adaptors using Cuadapt (0.9.4) and the following parameters: cutadapt -aAGTTGGATACCTGCAGTACTAGTCAGTGGGCCC -m16 -M16 -O33.  Trimmed mate-pairs were then aligned to the mm9 genome assembly using ELAND (CASAVA v1.8.1) with the following parameters: -ub Y\* --bam.  SAMtools was used to filter only reads with MAPQ ≥ 1 for downstream analysis.  Aligned reads were paired with mates and were filtered for PCR duplicates (>1 paired-end read with same start positions of both mates). Paired-end reads were categorized into interchromosomal, intrachromosomal (distance between mates > 5kb), and self (distance between mates < 5kb).  Only intrachromosomal reads with mates less than 1Mb apart and overlapping at least 1 Smc1a ChIP-seq peak were considered.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-seq and ChIA-PET samples are supplied with BED files containing coordinates of either ChIP-seq peaks or ChIA-PET interaction intervals. Wig files were generated for RNA-seq and ChIP-seq samples, normalized by sequencing depth, and converted to bigWig with the wigToBigWig utility.

source_name: Embryonic cortex

growth_protocol: Normal mouse gestation to day E10.5, E11.5, or E14.5

extract_protocol:
Total RNA was extracted with RNEasy kit (Qiagen) for RNA-seq. For each ChIP-Seq and ChIA-PET, limb bud tissue was crosslinked with 1% formaldehyde at room temperature and stored at -80°C. Chromatin was extracted and sheared by sonication. Soluble chromatin was combined Protein G Dynabeads prebound with appropriate antibodies at 4C overnight.  Beads were collected with magnet and washed 5 or 8x.  For ChIP-seq, chromatin was eluted with TE+1%SDS at 65C for 10 minutes.  Crosslinks were reversed at 65C overnight then chromatin was purified with PCR cleanup kit (Qiagen).     For ChIA-PET, chromatin was end-repaired, ligated to ChIA-PET adaptors, and the 5’ ends of the adaptors was phosphorylated.  Chromatin was eluted as described above, followed by ligation of chromatin complexes and reversal of crosslinking (65C overnight). DNA was purified with phenol:chloroform extraction and ethanol precipitation.
ChIP-seq libraries were constructed with the standard procedure included with the Illumina ChIP-Seq kit but paired-end adaptors (PE-102-1001) were used instead of those provided in the kit.  For multiplexed samples, Illumina Multiplexing adapters and primers (PE-400-1001) were used. RNA-seq Iibraries were constructed with the standard procedure included with the Illumina TruSeq RNA Sample Preparation kit.  ChIA-PET libraries were constructed with Illumina paired-end adaptors (PE-102-1001) and the protocol outlined in Fullwood, et al. 2010 (PMID:20069536)

treatment_protocol: Pregnant mothers were euthanized and embryos were removed and placed in cold PBS. Tissue (forelimbs, hindlimbs, cortex) was dissected with forcepts.

tag: embryonic day

value: H3K27me3 (Millipore, Cat. No. 07-449, Lot No. DAM1588246)

value: Embryonic cortex


title: CHD1_del

description: Monococcal nuclease treated chromatin profile for CHD1 deletion strain

source_name: CHD1 deletion strain

value: CHD1 deletion strain


title: Input_H3K27me3_e11.5cortex


title: ChIPSeq_H3K27ac_e14.5cortex

value: H3K27ac (Abcam, Cat. No. ab4729, Lot No. GR46123)


title: ChIPSeq_H3K27me3_e14.5cortex


title: Xrn1-TAP

description: yeast cells grown in YPD


title: RNA_seq_e11.5forelimb

molecule: polyA RNA

source_name: Forelimb bud

value: Forelimb bud


title: ChIPSeq_H3K4me2_e14.5cortex

value: H3K4me2(Millipore, Cat. No. 07-030, Lot No. DAM1724042)


title: ChIA_PET_Smc1a_e11.5limb_rep2

description: library strategy: ChIA-PET

source_name: Limb bud

value: Smc1a (Bethyl, Cat. No. A300-055A, Lot No. A300-055A-3)

value: Limb bud


title: ChIA_PET_Smc1a_e11.5limb_rep1

description: library strategy: ChIA-PET


title: RNA_seq_e11.5hindlimb

source_name: Hindlimb bud

value: Hindlimb bud


title: ChIPSeq_Smc1a_e14.5cortex


title: ctrl_WT_del_rep01

value: BY4741, MATa his3-del1 leu2-del0 met15-del0 ura3-del0

value: Wild-type control for deletion strains


title: SNF2_del

description: Monococcal nuclease treated chromatin profile for SNF2 deletion strain

source_name: SNF2 deletion strain

value: SNF2 deletion strain


title: TUP1_del_rep1

description: Monococcal nuclease treated chromatin profile for TUP1 deletion strain

source_name: TUP1 deletion strain

value: TUP1 deletion strain


title: 20120724_RPF-Seq_28hpf

description: size-selected on PAGE gel, ~30nt

source_name: Whole embryos

value: 28 hours post fertilization

value: TL/AB

value: ribosome footprinted RNA


title: ChIPSeq_H3K27me3_e11.5limb

value: H3K27me3 (Millipore, Cat. No. 07-449, Lot No. JCB1873477)


title: RPB2_tet

description: Monococcal nuclease treated chromatin profile for RPB2 Tet promotor-shutoff strain

source_name: RPB2 Tet promotor-shutoff strain

growth_protocol: For Tet promoter-shutoff strains, doxycyline was added at 10μg/ml for 24 h in order to obtain down-regulation for the gene of interest. Cells were then diluted to an OD600 of 0.2 and grown in the presence of doxycycline (10μg/ml) until mid-log phase.

value: BY4741, MATa his3-del1 leu2-del0 met15-del0 URA3::CMV-tTA

value: RPB2 Tet promotor-shutoff strain


title: MSI1_del_rep1

description: Monococcal nuclease treated chromatin profile for MSI1 deletion strain

source_name: MSI1 deletion strain

value: MSI1 deletion strain


title: Input_H3K27me3_e14.5cortex

value: Embryonic cortex


title: Input_H3K27ac_H3K4me2_e14.5cortex


title: ChIPSeq_H3K4me2_e11.5limb


title: Input_Smc1arep1_e11.5limb


title: ChIPSeq_CTCF_e10.5hindlimb_rep2

value: CTCF (Millipore, Cat. No. 07-729, Lot No. DAM1745366)


title: Input_H3K27me3_Smc1rep2_e11.5limb


title: Input_H3K4me2_e11.5limb


title: ChIPSeq_CTCF_e10.5hindlimb_rep1


title: ChIPSeq_CTCF_e10.5forelimb_rep2


title: ChIPSeq_Smc1a_e11.5limb_rep1


title: ChIPSeq_Smc1a_e11.5limb_rep2


title: ChIPSeq_CTCF_e10.5forelimb_rep1


title: ChIPSeq_CTCF_e11.5_limb

value: CTCF (Millipore, Cat. No. 07-729, Lot No. JCB1881349)


title: INK4A/ARF -/- mGIC BMI1

status: Public on May 21 2013

city: Berlin

first: Gaetano

last: Gargiulo

address: Robert-Rössle-Straße 10

department: Molecular Oncology

country: Germany

institute: Max Delbrüch Center

data_processing:
Alignment used BWA, and peak calling used MACS, and Clustering used http://bips.u-strasbg.fr
mm9 and hg19
Genome build: mm9

source_name: INK4A/ARF -/- mGIC BMI1

growth_protocol: Mouse cells were grown as in Bruggeman, 2007, human cells as in Pollard 2009

extract_protocol: Libraries were prepared according to Illumina's instructions. DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA, library fragments of ~230 bp (insert plus adaptor and PCR primer sequences) were selected from an agarose gel. The final size-selected lib. was amplified by 15 cycle of PCR followed by column purification of amplified DNA. The purified DNA was captured on an Illumina flow cell for cluster generation.   ChIP was performed by cross-linking proteins to DNA using 2mM DSG and 1% formaldehyde solution (Gargiulo et al, 2009). Immunoprecipitated DNA (ChIP-seq) and Input DNA were Libraries were sequenced on the Genome Analyzer of HiSeeq2000 following the manufacturer's protocols.

treatment_protocol: GBM1 were cultured in presence of Doxyclyne (48h), Arvanil (24h) and BMP7 (3h), only for the ATF3 ChIP-seq experiment.

value: mouse Glioma Initiating Cells

tag: genetic background

value: INK4a/Arf FVB+EgfrVIII (retroviral)

value: INK4A/ARF -/-

tag: chip antibody manufacturers

value: Millipore, Abcam, Abnova

tag: chip antibody catalog #'s

tag: chip antibody lot #'2


title: Input_CTCF_e11.5limb

value: Limb bud


title: INK4A/ARF -/- Astrocytes BMI1

source_name: INK4A/ARF -/- Astrocytes BMI1

value: Astrocytes

value: INK4a/Arf FVB


title: INK4A/ARF -/- mNPC 2 Input

source_name: INK4A/ARF -/- mNPC 2 Input

value: Adult Neural Progenitor cells


title: PA1_2

first: Tuan Zea

last: Tan

address: 14 Medical Drive, #11-01, Singapore

department: Cancer Science Institute Singapore

institute: National University of Singapore

description: replicate

data_processing:
library strategy: DNA-seq
Basecalls performed using CASAVA version 1.6
DNA-seq reads were aligned to the shRNA TRC1.0 library using Bowtie version0.12.7 with options --solexa1.3-quals –n 0 –l 5 -v 0 -k 1 -m 1 --best –strata -y –nomaqround
Mapped reads are counted for each shRNA hairpins
Genome_build: shRNA library TRC1.0 (http://www.broadinstitute.org/rnai/trc/lib)
Supplementary_files_format_and_content: tab-delimited text files include shRNA counts for each Sample.

source_name: Cultured ovarian cancer cell line

extract_protocol:
Genomic DNA was harvested from the resulting cells by PureLink Genomic DNA kits (#K1820-01, Invitrogen)
Amplified DNA (20 ng) from PCR was used to construct a sequencing library using a ChIP-Seq sample preparation kit (#IP-102-1001, Illumina, San Diego, CA). The two sample-multiplexing sequencing method was used individually, with multiplexing index 6 and index 12 primers for each sample, (Illumina, #PE-400-1001).

tag: cell line

value: ovarian cancer cells


title: PA1_3

description: replicate


title: INK4A/ARF -/- mNPC 2 BMI1

source_name: INK4A/ARF -/- mNPC 2 BMI1


title: HEYA8_3

description: replicate


title: OVCA433_3

description: replicate


title: OVCA433_1

description: replicate


title: OVCA433_2

description: replicate


title: HEYA8_2

description: replicate


title: Input_Smc1a_e14.5cortex

tag: tissue


title: PEO1


title: HEYA8_1

description: replicate


title: PA1_1

description: replicate


title: A1847


title: HEYC2


title: SKOV6


title: PA1


title: SKOV3


title: HEY

value: HEY


title: OVCA433


title: OVCAR8


title: OVCA429


title: HEYA8


title: SKOV4


title: HuR

city: Baltimore

first: Adam

middle: D

last: Ewing

state: MD

address: 733 N. Broadway, MRB 439

department: Genetic Medicine

institute: Johns Hopkins University

data_processing:
Remove Adapter: cutadapt -a GATCTCGTATGCCGTCTTCTGCTTG in.fastq > out.fastq
Align to reference genome: bowtie2 -x <indexed reference>  -U in.fastq -S out.sam --quiet -p 3 -D 15 -R 2 -L 20 -N 1 --rdg 20,20 --rfg 20,20
Convert SAM to BAM (samtools view -b)
Select all alignments with a T-to-C change relative to the top strand
Convert BAM to pileup (samtools mpileup)
Normalize pileup depth by number of aligned reads in sample
Convert mpileup output to wiggle (wig): http://genome.ucsc.edu/goldenPath/help/wiggle.html
Convert wiggle to bigWig (wigToBigWig)
Genome_build: GRCh37 (UCSC hg19)
Supplementary_files_format_and_content: aligned sequence depth in bigWig (.bw) format: http://genome.ucsc.edu/goldenPath/help/bigWig.html

growth_protocol: HEK293T cells were maintained in a tissue culture incubator at 5% CO2, 370C in high glucose Dulbecco’s modified Eagle medium (DMEM) without pyruvate  (Invitrogen) supplemented with 10% fetal bovine calf serum, 2 mM L-glutamine and 100U/ml Penicillin-Streptomycin.

extract_protocol:
UV-irradiated cells were lysed, treated with RNAseT1, and the RNA-protein complex was immunoprecipitated with anti-FLAG agarose beads for 1 hour at 40C. RNA was labeled with gamma ATP and the crosslinked RNA-proteins were resolved in a SDS-PAGE gel. ORF1 and HuR RNA-protein complexes were cut from the gel.
Recovered RNA was converted into cDNA libraries using a small RNA cloning protocol described in Hafner et al (Methods, 2008, doi: 10.1016/j.ymeth.2007.09.009)

treatment_protocol: The PAR-CLIP protocol was performed as described in Hafner et al. (Cell 2010, doi: 10.1016/j.cell.2010.03.009) with minor modification. HEK293T cells were transiently transfected with plasmid constructs and grown for 24 hours before adding 100 uM 4-SU and grown for another 12 hrs.


title: ORF1p replicate 2

description: library strategy: PAR-CLIP


title: ORF1p replicate 1

description: library strategy: PAR-CLIP


title: A2780J


title: ORF1p/RNP replicate 2

description: library strategy: PAR-CLIP


title: CH1


title: hGBM 1 Input (BMI1)

data_processing:
Alignment used BWA, and peak calling used MACS, and Clustering used http://bips.u-strasbg.fr
mm9 and hg19
Genome build: hg19

source_name: hGBM 1 INPUT (BMI1)

value: Glioblastoma Multiforme stem like

value: Multiple genetic aberrations


title: hGBM1 + Dox + Arvanil + BMP7 Input (ATF3)

source_name: hGBM1 + Dox + Arvanil + BMP7 INPUT (ATF3)

tag: treatment

value: Dox + Arvanil + BMP7


title: hGBM1 + Dox + Arvanil + BMP7 ATF3

source_name: hGBM1 + Dox + Arvanil + BMP7 ATF3

tag: chip antibody manufacturer

value: Abcam

tag: chip antibody catalog #

tag: chip antibody lot #


title: hGBM 1 BMI1

source_name: hGBM 1 BMI1

tag: cell type

tag: chip antibody


title: hGBM 5 BMI1

source_name: hGBM 5 BMI1


title: INK4A/ARF -/- mNPC 1 Input

source_name: INK4A/ARF -/- mNPC 1 Input


title: hNPCs BMI1

source_name: hNPCs BMI1

value: Foetal Neural Progenitor cells

value: Normal; diploid


title: hNPCs Input

source_name: hNPCs INPUT


title: ORF1p/RNP replicate 1

description: library strategy: PAR-CLIP


title: INK4A/ARF -/- mNPC 1 BMI1

source_name: INK4A/ARF -/- mNPC 1 BMI1


title: 32-cell.TABseq

status: Public on May 20 2013

city: Beijing

first: Lan

last: Jiang

address: NO.1 Beichen West Road, Chaoyang District

country: China

institute: Beijing Institute of Genomics

data_processing:
Library strategy: TAB-Seq
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality
Zv9 reference genome was downloaded from UCSC. The 48,502 bp Lambda genome was also included in the reference sequence as an extra chromosome for calculating bisulfite conversion rate. Filtered paired-end methylC-seq reads were mapped against the reference by Bismark_v0.6.4 (Krueger and Andrews, 2011) with stringent parameters:  -n 2 -l 60  -e 100 -X 600. A custom script was used to examine whether pair-end reads were overlapped and the overlapped part is trimmed from one end to prevent counting twice from the same observation.
For CpG i, define mi as the number of reads showing methylation over position i (both strands). Define ui as the number of reads showing lack of methylation over CpG i. The methylation level is estimated as mi/( mi + ui), which is an estimate of the probability that CpG i is methylated in a molecule sampled randomly from the cell population. Because CpG methylation is symmetric, mi and ui include observations associated with the cytosines on both strands for the i-thCpG.
for TAB-seq , 5-hmC mapping was performed as described (Yu et al., 2012). Paired-reads were mapped uniquely to the reference genome (Zv9, USCS) plus Lambda and pUC19 DNA sequence as extra chromosome by Bismark. All the CpG sites disrupted by SNPs were filtered from the analysis. 5hmC was called with a binomial distribution follows the method previously reported (Yu et al., 2012). A binomial distribution (Lister et al., 2009)was applied to model this probabilistic event with N as the depth of sequencing at the cytosine and p as the 5mC non-conversion rate. Efficient conversion of unmodified cytosine to uracil and efficient conversion of 5mC to 5caU/U were calculated by spiked M. SsI treated lambda DNA.
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: wig files contain  methylaion level of each CpG site

extract_protocol:
TAB-seq:5-hmC library was constructed as described (Yu et al., 2012). Briefly,fragmented genomic DNA  with spike-in controls was ligated to methylated  adapter , and was firstly glucoslyated and oxidized before bisulfite treatment. Efficient conversion of unmodified cytosine to uracil and efficient conversion of 5mC to 5caU/U were calculated by spiked M. SsI treated lambda DNA. 5hmc protection rate was calculated by spiked pUC19 DNA .
Sperms were released by gently but repeatedly disrupting the testis with a pipette tip in HBSS. Unfertilized oocytes were collected by squeezing the abdomen of females about 5 minutes after spawning. Embryos were grown in embryo medium at 28°C, staged according to standard morphological criteria..Zebrafish embryos were chilled on ice, washed by pre-chilled PBS buffer three times to avoid the contamination and homogenized in 10 vol buffer N (10 mM HEPES, pH 7.5, 250 mM sucrose, 50 mMNaCl, 5 mM MgCl2, 10 µg/ml cytochalasin B, 1 mMdithiothreitol and protease inhibitors) with ten strokes of a loose fitting pestle in a glass homogenizer, and lysates were decanted for 20 min on ice. Then lysates were filtered with 70µm cell strainer（BD Biosciences) and centrifuged through 1 M sucrose at 1000 g for 30 min. Pelleted nuclei from embryos were subjected to genomic DNA extraction by standard phenol precipitation method.

tag: developmental stage

value: embryo

tag: stain

value: Tubingen

tag: library strategy

value: TAB-Seq


title: testis.BSseq

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality
Zv9 reference genome was downloaded from UCSC. The 48,502 bp Lambda genome was also included in the reference sequence as an extra chromosome for calculating bisulfite conversion rate. Filtered paired-end methylC-seq reads were mapped against the reference by Bismark_v0.6.4 (Krueger and Andrews, 2011) with stringent parameters:  -n 2 -l 60  -e 100 -X 600. A custom script was used to examine whether pair-end reads were overlapped and the overlapped part is trimmed from one end to prevent counting twice from the same observation.
For CpG i, define mi as the number of reads showing methylation over position i (both strands). Define ui as the number of reads showing lack of methylation over CpG i. The methylation level is estimated as mi/( mi + ui), which is an estimate of the probability that CpG i is methylated in a molecule sampled randomly from the cell population. Because CpG methylation is symmetric, mi and ui include observations associated with the cytosines on both strands for the i-thCpG.
for TAB-seq , 5-hmC mapping was performed as described (Yu et al., 2012). Paired-reads were mapped uniquely to the reference genome (Zv9, USCS) plus Lambda and pUC19 DNA sequence as extra chromosome by Bismark. All the CpG sites disrupted by SNPs were filtered from the analysis. 5hmC was called with a binomial distribution follows the method previously reported (Yu et al., 2012). A binomial distribution (Lister et al., 2009)was applied to model this probabilistic event with N as the depth of sequencing at the cytosine and p as the 5mC non-conversion rate. Efficient conversion of unmodified cytosine to uracil and efficient conversion of 5mC to 5caU/U were calculated by spiked M. SsI treated lambda DNA.
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: wig files contain  methylaion level of each CpG site

source_name: testis

extract_protocol:
MethylC-Seq:Genomic DNA (>100ng) spiked with 0.5％ unmethylated cl857 Sam7 Lambda DNA (Promega) was sonicated into 100-500bp fragments with Covaris S2 (Applied Biosystems). Fragmented DNA was then subjected to end repair with the end repair enzyme mix (NEB), dA tailing with the Klenow 3´-5´ exo- (NEB) and ligation of methylated multiplexing adapters (Illumina) using T4 DNA ligase (NEB). Adapter-ligated DNA of 280-500bp was purified by 2％agarose gel electrophoresis, and bisulfite conversion was performed using the EZ DNA methylation-Gold kit (Zymo Research) according to the instruction manual.
Sperms were released by gently but repeatedly disrupting the testis with a pipette tip in HBSS. Unfertilized oocytes were collected by squeezing the abdomen of females about 5 minutes after spawning. Embryos were grown in embryo medium at 28°C, staged according to standard morphological criteria..Zebrafish embryos were chilled on ice, washed by pre-chilled PBS buffer three times to avoid the contamination and homogenized in 10 vol buffer N (10 mM HEPES, pH 7.5, 250 mM sucrose, 50 mMNaCl, 5 mM MgCl2, 10 µg/ml cytochalasin B, 1 mMdithiothreitol and protease inhibitors) with ten strokes of a loose fitting pestle in a glass homogenizer, and lysates were decanted for 20 min on ice. Then lysates were filtered with 70µm cell strainer（BD Biosciences) and centrifuged through 1 M sucrose at 1000 g for 30 min. Pelleted nuclei from embryos were subjected to genomic DNA extraction by standard phenol precipitation method.

value: testis

value: organ

value: BS-Seq


title: 1k-cell.BSseq


title: 64-cell.BSseq


title: 32-cell.BSseq


title: 16-cell.BSseq


title: egg.BSseq

source_name: egg

value: egg

value: gamete


title: sperm.BSseq

source_name: sperm

value: sperm


title: input.5hmc.hMeDIP

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality
FASTQ sequence files were aligned to the Zebrafish reference (Zv9) using BWA Version: 0.6.1-r104, retaining only unique, non-duplicate genomic matches with no more than 2 mismatches within the first 32bp.
5-hmC peaks were identified using MACS with the following parameters: effective genome size = 1.40e+09; band width = 200; Tag size = 101; P-value cutoff = 1.00e-05; model fold= 4,20; ranges for calculating regional lambda are: peak_region, 200, 1000.
all the processed files are  the raw results  got  from  MACS without any filter step.
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: tab-delimited text files include peak location for each Sample …

source_name: embryo

extract_protocol:
Zebrafish embryos were chilled on ice, washed by pre-chilled PBS buffer three times to avoid the contamination and homogenized in 10 vol buffer N (10 mM HEPES, pH 7.5, 250 mM sucrose, 50 mM NaCl, 5 mM MgCl2, 10 µg/ml cytochalasin B, 1 mM dithiothreitol and protease inhibitors) with ten strokes of a loose fitting pestle in a glass homogenizer, and lysates were decanted for 20 min on ice. Then lysates were filtered with 70µm cell strainer（BD Biosciences) and centrifuged through 1 M sucrose at 1000 g for 30 min. Pelleted nuclei from embryos were subjected to genomic DNA extraction by standard phenol precipitation method.
5-hmC contained DNA were enriched as described(Robertson et al., 2012) with modifications. Fragmented DNA was then subjected to end repair with the end repair enzyme mix (NEB), dA tailing with the Klenow 3´-5´ exo- (NEB) and ligation of multiplexing adapters (Illumina) using T4 DNA ligase (NEB). Adapter-ligated DNA of 280-500bp was purified by 2％ agarose gel electrophoresis, and then 5hmC was converted to β-glucosyl-5-hmC (β-glu-5-hmC) by T4 β-glucosyltransferase. β-glu-5-hmC contained DNA fragment was further pulled down by J-binding protein 1 (JBP1)-coated magnetic beads from Quest 5-hmC™ DNA Enrichment Kit (zymo research). Finally, enriched DNA fragments that have adapters on both ends were amplified using PCR to create the final sequencing library and sequenced by Illumina HiSeq 2000. A quantity of fragmented genomic DNA was set aside as input DNA. The efficiency of precipitation was checked by kit included positive control fragments.


title: hGBM 5 Input

source_name: hGBM 5 INPUT

tag: genotype


title: 1kcell.5hmc.hMeDIP


title: sperm.RNA-seq

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality
FASTQ sequence files were aligned to the Zebrafish reference (Zv9) using BWA Version: 0.6.1-r104, retaining only unique, non-duplicate genomic matches with no more than 2 mismatches within the first 32bp.
Using uniquely mapped reads, read counts per exon were obtained, followed by summarizing the read counts for all exons within a gene for all genes in UCSC Zv9 refFlat table. For each sample, RPKM was computed as the number of reads which map per kilobase of exon model per million mapped reads for each gene.
As there are global shifts in the levels of mRNA during embryonic development(Aanes et al., 2011). TMM (Trimmed Mean of M values)(Robinson and Oshlack, 2010) method implemented in R package edgeR was used to normalize the global shifts in gene expression, resulting in a shifting factor (TMM factor) for each sample. Briefly, the relative abundance of each transcript was estimated as the number of reads for transcript i divided by the total number of reads in sample j. Eij=Rij/Rj, where Eij is the relative abundance of transcript i in sample j, Rij is the number of reads for gene i in sample j, and Rj is the total number of reads in sample j. The average Rj for all samples were summed to generate a common library size: Xavg = sum(R1~Rn)/n. A pseudo library size were calculated by multiplying Xavg with the TMM factor (Xj = Xavg*Zj), where Zj was TMM factor computed as described above. These pseudo read libraries were reassigned to each gene based on previously estimated Eij value to get the normalized dataset (normEij = Eij*Xj).
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: tab-delimited text files include reads count of all exons within a gene for all genes, which is TMM-normalized among sperm ,egg , 1kcell and germring samples.

extract_protocol:
Zebrafish embryos were chilled on ice, washed by pre-chilled PBS buffer three times to avoid the contamination and homogenized in 10 vol buffer N (10 mM HEPES, pH 7.5, 250 mM sucrose, 50 mMNaCl, 5 mM MgCl2, 10 µg/ml cytochalasin B, 1 mMdithiothreitol and protease inhibitors) with ten strokes of a loose fitting pestle in a glass homogenizer, and lysates were decanted for 20 min on ice. Then lysates were filtered with 70µm cell strainer（BD Biosciences) and centrifuged through 1 M sucrose at 1000 g for 30 min. Pelleted nuclei from embryos were subjected to genomic DNA extraction by standard phenol precipitation method. Total RNA was extracted from homogenized whole embryos using Trizol (Invitrogen) according to manufacturer's instructions.
The RNA-seq libraries were constructed as per the TruSeq RNA Sample Preparation Guide (Illumina). Briefly, mRNA was purified using poly-T oligo-attached magnetic beads and fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were then reverse transcribed into first strand cDNA, followed by second strand cDNA synthesis to generate double-stranded (ds) cDNA. After end repair and dA tailing, multiple indexing adapters were ligated to the ends of the double strand cDNA. The ligation products were size selected to contain 350-450bp fragments and purified. Finally, DNA fragments that have adapters on both ends were enriched using PCR to create the final sequencing library and sequenced by Illumina HiSeq 2000.

tag: devolopmetal stage


title: germring.RNA-seq

value: germ ring


title: 16cell.5hmc.hMeDIP


title: hGBM 1 BMI1 REP 2

source_name: hGBM 1 BMI1 REP 2

value: Millipore, Abcam, Abnova


title: 1kcell.RNA-seq


title: egg.RNA-seq


title: 2cell.5hmc.hMeDIP


title: sperm.5hmc.hMeDIP.


title: Germring.BSseq

source_name: Germring

value: Germring


title: AX4 grown on Bs, replicate 2

city: Houston

first: Gad

last: Shaulsky

state: TX

address: One Baylor Plaza

department: Molecular and Human Genetics

institute: Baylor College of Medicine

description:
AX4_Bs_bio2
mRNA

data_processing:
The data was mapped to the D. discoideum genome with bowtie version 0.12.7. Read qualities were used. Other parameters: seed length = 28, allowed mismatches = 2, allowed up to 1 alignment per read, with bowtie options -a, --strata, --best. Unmapped reads were trimmed from 3' by 2 nucleotides, which was repeated 5 times.
Raw expression values were computed as the number of reads that were uniquely mapped to the exons of genes.
Normalized expression were scaled by mappable exon lengths. This normalization is similar to the RPKM normalization (Reads Per Kilobase of exon per Megabase of library size), but instead of dividing by exon lengths we used the length of uniquely mappable parts of the exon. To obtain uniquely mappable parts, all possible subsequences of the reference genome (of the same length as reads in raw data) are mapped back to the reference genome, obtaining the number of uniquely mapped sequences to the exons (Exon_mappable). As a library size we used the total number of all uniquely mapped reads from the experiment, excluding the non-polyadenylated genes (N_unique). Normalized expressions were computed as follows: Exp = 10^9*raw/(N_unique * Exon_mappable).
D. discoideum Chromosome: 1,2,3,4,5,6,M, and floating contigs (created: 05-13-2009 13:53) from the DictyBase (chromosomes 1,2,3,4,5,6 and mitochondrial DNA are the same as on NCBI assembly "dicty_2.7"). Regions [3016085, 3768655] from chr2, [64985, 72996] from chrBF and [42801, 78150] from chrR were masked.
Genome_build: dicty_2.7
Supplementary_files_format_and_content: Processed data files are tab-separated files with two columns: the first containing gene names and the second its expression. Files ending with "_expr_raw.tab" contain raw expression values, while files ending with "_expr_norm.tab" contain normalized expression values.

organism: Dictyostelium discoideum

source_name: AX4 grown on Bs

growth_protocol: D. discoideum was recovered on K. pneumoniae bacterial lawn on SM medium, then grown axenically on HL5 medium and maintained at mid-log phase (2.5x10^6 cells/ml) before subsequent experiments.

extract_protocol:
2.5x10^7 washed amoebae cells were immediately lysed in TRIZOL® reagent, and total RNA was prepared according to the manufacturer's protocol (Invitrogen by Life Technologies).
We processed 20 μg of total RNA through one round of poly-A selection, RNA fragmentation, first-strand and second-strand cDNA synthesis. Two biological replicates were analyzed for each condition. We sequenced the cDNA libraries (read length = 35 bases) on a high-throughput Illumina Genome Analyzer II using the manufacturer's recommended pipeline (versions 1.2 and 1.3).

treatment_protocol: 1-3x10^7 D. discoideum cells were mixed with 2 ml of thick bacterial culture and spread evenly on a 10-cm petri plate of buffered agar. After 14-16 hours, amoebae and bacteria were harvested and resuspended in ice-cold mSor buffer to wash away bacteria by low centrifugation (200xg at 4oC). Supernatant with bacteria was discarded. This procedure was repeated 2-4 times until no visible bacteria were detected in supernatant.

tag: strain

tag: growth on bacteria

value: Bacillus subtilis

tag: replicate


title: AX4 grown on Pa, replicate 2

description:
AX4_Pa_bio2
mRNA

source_name: AX4 grown on Pa

value: Pseudomonas aeruginosa


title: AX4 grown on Bs, replicate 1

description:
AX4_Bs_bio1
mRNA


title: AX4 grown on Sa, replicate 1

description:
AX4_Sa_bio1
mRNA

source_name: AX4 grown on Sa

value: Staphylococcus aureus


title: AX4 grown on Sa, replicate 2

description:
AX4_Sa_bio2
mRNA


title: AX4 grown on Kp, replicate 2

description:
AX4_Kp_bio2
mRNA

source_name: AX4 grown on Kp

value: Klebsiella pneumoniae


title: AX4 grown on Kp, replicate 1

description:
AX4_Kp_bio1
mRNA


title: 128-cell.BSseq


title: AX4 grown on Pa, replicate 1

description:
AX4_Pa_bio1
mRNA


title: 2_hAgo1_sRNA_replicate2

city: Cold Spring Harbor

first: Astrid

last: Haase

state: NY

address: One Bungtown Road

institute: Cold Spring Harbor Laboratory

description: small 5' monophosphorylated RNAs (19-29nt size selected) associated with recombinant hAgo1 protein purified from SF9 cells

data_processing:
fastq to fasta conversion
Clipping 3' Adapter: TGGAATTCTCGGGTGCCAAGGC Min. Length: 15; Non-Clipped reads discarded
length distribution
filter 20nt reads
first nucleotide distribution of 20nt reads
Supplementary_files_format_and_content: tabular file of filtered reads with length==20

organism: Spodoptera frugiperda

extract_protocol:
RNA extraction using TRIZOL reagent followed by size fractionation using PAGE purification (19-29nt)
libraries were prepared according to Brennecke et al., 2007
small RNA library preparation by linker ligation (dependent on 5' monophosphate)

value: SF9 cell line

tag: protein


title: 5_hAgo2_sRNA_replicate1

description: small 5' monophosphorylated RNAs (19-29nt size selected) associated with recombinant hAgo2 protein purified from SF9 cells


title: 1_hAgo1_sRNA_replicate1

source_name: recombinant hAgo1 protein purified from SF9 cells


title: 8_SF9input(hAgo1)_sRNA_replicate2

description: small 5' monophosphorylated RNAs (19-29nt size selected) from SF9 cell infected baculovirus expressing hAgo1

source_name: SF9 cell infected with baculovirus expressing hAgo1

value: (input)


title: 6_hAgo2_sRNA_replicate2

source_name: recombinant hAgo2 protein purified from SF9 cells


title: 4_hAgo3_sRNA_replicate2

description: small 5' monophosphorylated RNAs (19-29nt size selected) associated with recombinant hAgo3 protein purified from SF9 cells


title: 3_hAgo3_sRNA_replicate1

source_name: recombinant hAgo3 protein purified from SF9 cells


title: Molecular barcode single cell MB3

status: Public on May 19 2013

city: New York City

first: Rahul

last: Satija

address: 101 Avenue of the Americas

institute: New York Genome Center

data_processing:
We created a Bowtie index based on the UCSC knownGene (8) transcriptome, and aligned paired-end reads directly to this index using Bowtie v 0.12.7 with command line options -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -a -m 200.
Next, we ran RSEM v1.11 with default parameters on these alignments to estimate expression levels. RSEM’s gene level expression estimates (tau) were multiplied by 1,000,000 to obtain transcript per million (TPM) estimates for each gene.
Genome_build: mm9
Supplementary_files_format_and_content: File allGenesTPM.txt represents a matrix of gene expression estimates across all non-MolecularBarcode samples. File umbExp.txt represents a matrix of gene expression estimates across all MolecularBarcode samples. Linked as supplementary files on Series record.

source_name: BMDC (4h LPS stim)

growth_protocol: Cells were cultured and stimulated with LPS as previously described (Amit et. al 2009)

extract_protocol:
cDNA from each sample was prepared using the SMARTer cDNA synthesis (Clontech). RNA libraries were prepared for sequencing using standard Illumina protocols.
For the three molecular barcode (MB) libraries, we used a slightly modified SMARTer oligo which contained a 4bp random molecular barcode (NNNN) immediately after the SMARTer sequence.

value: Bone Marrow-derived Dendritic Cell (BMDC)

value: LPS-stimulation

tag: cell count

value: 1 cell

tag: protocol

value: molecular barcodes (MB) using a modified SMARTer protocol


title: dme_gd144_input_2to4h_1

description: input DNA from gd7 embryos with one copy of a 25kb TwistBAC where the twi promoter has been replaced by the snail core promoter and the twi coding region replaced by the yellow sequence (Bac144)

data_processing:
Reads were aligned to the UCSC dm3 genome with the addition of specific BAC sequences as pseudo-chromosomes with bowtie v0.12.7. Only uniquely aligning reads with up to two mismatches were retained. For paired-end samples, successfully aligned mate pairs were joined to a single fragment.
Genome Build:
gd144_input.bed: dm3

source_name: input DNA from gd7 embryos expressing the transgene twiBAC-snaPr-Yellow

tag: tissue

value: whole gd7 embryos

tag: tissue type

value: dorsal ectoderm

value: maternally gd7/gd7 mutants and zygotically expressing the transgene twiBAC-snaPr-Yellow

value: none (input)


title: Molecular barcode single cell MB2

tag: treatment


title: 10,000 cell population P3

data_processing:
We created a Bowtie index based on the UCSC knownGene (8) transcriptome, and aligned paired-end reads directly to this index using Bowtie v 0.12.7 with command line options -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -a -m 200.
Next, we ran RSEM v1.11 with default parameters on these alignments to estimate expression levels. RSEM’s gene level expression estimates (tau) were multiplied by 1,000,000 to obtain transcript per million (TPM) estimates for each gene.
Genome_build: mm9
Supplementary_files_format_and_content: File allGenesTPM.txt represents a matrix of gene expression estimates across all non-MolecularBarcode samples. File umbExp.txt represents a matrix of gene expression estimates across all MolecularBarcode samples.  Linked as supplementary files on Series record.

extract_protocol:
cDNA synthesis and amplification:
We used the SMARTer Ultra Low RNA Kit (Clontech, Mountain View, CA) to prepare amplified cDNA. We added 1   l of 12   M 3' SMART primer (5'   AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N (N = A, C, G, or T; N-1 = A, G, or C)), 1   l of H2O, and 2.5   l of Reaction Buffer onto the RNA capture beads. We mixed them well by pipetting, heated the mixture at 72  C for 3 minutes and placed it on ice. First-strand cDNA was synthesized with this RNA primer mix by adding 2   l of 5x first-strand buffer, 0.25   l of 100mM DTT, 1   l of 10 mM dNTPs, 1   l of 12   M SMARTer II A Oligo (5'   AAGCAGTGGTATCAACGCAGAGTACXXXXX (X = undisclosed base in the proprietary SMARTer oligo sequence)), 100 U SMARTScribe RT, and 10 U RNase Inhibitor in a total volume of 10   l and incubating at 42  C for 90 minutes followed by 10 minutes at 70  C. We purified the first strand cDNA by adding 25   l of room temperature AMPure XP SPRI beads (Beckman Coulter Genomics, Danvers, MA), mixing well by pipetting, incubating at room temperature for 8 minutes. We removed the supernatant from the beads after a good separation was established. We carried out all of the above steps in a PCR product   free clean room. We amplified the cDNA by adding 5   l of 10x Advantage 2 PCR Buffer, 2   l of 10 mM dNTPs, 2   l of 12   M IS PCR primer (5'    AAGCAGTGGTATCAACGCAGAGT), 2   l of 50x Advantage 2 Polymerase Mix, and 39   l H2O in a total volume of 50   l. We performed the PCR at 95  C for 1 minute, followed by 21 cycles of 15 seconds at 95  C, 30 seconds at 65  C and 6 minutes at 68  C, followed by another 10 minutes at 72  C for final extension. We purified the amplified cDNA by adding 90   l of AMPure XP SPRI beads and washing with 80% ethanol.
We created Illumina sequencing libraries from this amplified cDNA using standard protocols.
cDNA shearing and library construction:
We added the purification buffer (Clontech) to the amplified cDNA to make a total volume of 76   l. We sheared the cDNA in a 100   l tube with 10% Duty Cycle, 5% Intensity and 200 Cycles/Burst for 5 minutes in the frequency sweeping mode (Covaris S2 machine, Woburn, MA). We purified the sheared cDNA with 2.2 volumes AMPure XP SPRI beads.
We prepared indexed paired-end libraries for Illumina sequencing as described (J. Z. Levin et al., Nature Methods 7, 709 (2010)., with the following modifications. First, we used a different indexing adaptor (containing an 8-base barcode) for each library. Second, we size-selected the ligation product by using two rounds of 0.7 volume of AMPure XP SPRI bead cleanup with the first round starting volume at 100   l. Third, we performed PCR with Phusion High-Fidelity DNA polymerase with GC buffer and 2 M betaine. Fourth, we used 55  C as the annealing temperature in PCR with the universal indexing primers (forward primer 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC, reverse primer 5'-CAAGCAGAAGACGGCATACGAGAT). Fifth, we performed 12 cycles of PCR. Sixth, we removed PCR primers using two rounds of 1.0 volume of AMPure beads.


title: 10,000 cell population P2


title: Single cell S18


title: Single cell S16


title: Single cell S17


title: Molecular barcode single cell MB1


title: Single cell S15


title: Single cell S14


title: 7_SF9input(hAgo1)_sRNA_replicate1


title: Single cell S12


title: Single cell S11


title: Single cell S9


title: Single cell S13


title: Single cell S4


title: Single cell S5


title: Single cell S6


title: Single cell S8


title: Single cell S3


title: Single cell S1


title: Single cell S2


title: PPARbeta-null

status: Public on May 18 2013

city: sacramento

first: HUIXIN

last: LIU

state: CA

address: 2 quay court apt 98

institute: UCD medical center

data_processing:
Illumina Casava1.7 software used for basecalling.
Total reads of RNA-sequencing experiments were analyzed using the combination of TopHat (2.0.0) and Cufflinks (1.3.0). TopHat was used to align reads to the mouse reference genome (NCBI37, mm9) and discover transcript splice sites. These alignments from TopHat were then assembled into transcripts using Cufflinks. Cuffdiff, a component of the Cufflinks package, was used to estimate FPKM (fragments per kilobase of exon model per million mapped fragments) and differentially express transcripts.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...

source_name: liver

growth_protocol: Wild-type (WT) and PPARβ-null mice (KO) male mice (3-5 month, C57BL/6) were kept in steel microisolator cages at 22°C with a 14-hr/10-hr light/dark cycle. Food and water were provided ad libitum throughout the entire feeding period.

extract_protocol:
Livers 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 2 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Mouse liver RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and integrity were determined by the Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis Chip. RNA-sequencing library preparation and sequencing was carried out by the Genome Sequencing Facility at University of Kansas Medical Center (Kansas City, KS). cDNA libraries were prepared with 2 µg of total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). The libraries were clustered and sequenced on an Illumina HiSeq 2000 instrument with 100 bp paired end reads.

value: tissue

tag: age

value: 4 month

value: PPARbeta-null


title: WT

value: wild type


title: 10,000 cell population P1

tag: cell type


title: Mbnl3 KO, biological rep3 (HITS-CLIP)

city: La Jolla

first: Ranjan

last: Batra

state: California

address: 2880 Torrey Pines Scenic Drive

department: Cellular and Molecular Medicine

institute: University of California San Diego

description: Protein-RNA complexes

data_processing:
Raw reads obtained from the Illumina pipeline included a 4 nt barcode (3 random positions and a G at the 4th position) at the 5 end followed by the CLIP tag and the 3 adaptor sequence. Results were filtered to remove low quality reads by requiring a minimum score of 20 in barcode positions and an average score of 20 in the following 25 nucleotides.
The filtered reads were then aligned to the mouse reference genome (mm9) by novoalign
Potential PCR duplicates, as judged from the starting position of genomic mapping and the barcode sequences, were removed to identify unique CLIP tags
Genome_build: mm9
Supplementary_files_format_and_content: BED

source_name: BL6 Mbnl3 ΔE2/ΔE2 Forelimb

growth_protocol:
C2C12 cells were grown in DMEM containing 20% FBS.
Forelimb dissected from Mbnl3 ΔE2/ΔE2 mice or WT female mice, E15 (15 dpc)

extract_protocol:
E15 forelimbs were dissected from wild type and Mbnl3 mutant BL6 mice, snap frozen in liquid nitrogen, ground to a fine powder, and crosslinked with UV-light in stratalinker 1800.
Three biological replicates were analyzed, each consisting of four forelimbs from two embryos. For C2C12 cells, five 10 cm plates per biological replicate, were UV-crosslinked followed by scraping cells in ice-cold PBS, centrifugation and freezing of cell pellets. For immunoprecipitation, protein lysates were denatured by adding SDS to a final concentration of 1% followed by heating (100°C/5 min) and subsequent dilution to a final SDS concentration of 0.1% (in lysis buffer). The Mb3/7 anti-Mbnl3 antibody (5 µg) and RNase A (concentrations of 55 U/ml and 0.55 U/ml for high and low RNase, respectively) were used and cDNA libraries were generated using RNA linkers and primers described for Ago CLIP.

value: Forelimb

tag: genotype/variation


title: Mbnl3 KO, biological rep2 (HITS-CLIP)

description: Protein-RNA complexes


title: Mbnl3 KO, biological rep1 (HITS-CLIP)

description: Protein-RNA complexes


title: WT, biological rep2 (HITS-CLIP)

description: Protein-RNA complexes

source_name: BL6 wild-type Forelimb

value: WT


title: Single cell S10


title: C2C12, biological rep3 (HITS-CLIP)

description: Protein-RNA complexes

source_name: C2C12 cells

value: DMEM + 20%FBS

tag: cell line


title: Single cell S7


title: WT, biological rep3 (HITS-CLIP)

description: Protein-RNA complexes


title: Mbnl3 KO, biological rep 2

data_processing:
Raw sequence reads were mapped back to the mouse reference genome (mm9) together with a comprehensive database of annotated exon junctions compiled from mouse, human, and rat mRNA/EST data.  The program tophat (v1.3.2) with bowtie (v3.4.6) was used for alignment (parameters: -a 5 -F 0.05 -r 150 -j <junction.hmr.raw>)
After read mapping, transcript structure between the sequenced ends could be ambiguous due to alternative splicing, and if this occurs, the structure was inferred using a bayes method by modeling the size distribution of cDNA fragments
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph format, with the four columms representing chromosome, start (0-based), end (1-based), and normalized read coverage (FPKM)

source_name: Mbnl3 ΔE2/ΔE2 Forelimb

growth_protocol: Forelimb dissected from Mbnl3 ΔE2/ΔE2 mice or WT female mice, E15 (15 dpc)

extract_protocol:
Embryos were genotyped and forelimbs were dissected from 3 mutant and 3 wild-type animals. Total RNA was extracted using Trizol and samples were purified with RNeasy™  Kit (Qiagen).
Sequencing libraries were prepared after PolyA RNA isolation using mRNA-sequencing kit (Illumina).

tag: gender

value: Female


title: C2C12, biological rep1 (HITS-CLIP)

description: Protein-RNA complexes


title: Mbnl3 KO, biological rep 1


title: WT, biological rep 3

source_name: wild-type Forelimb


title: Mbnl3 KO, biological rep 3


title: C2C12, biological rep2 (HITS-CLIP)

description: Protein-RNA complexes


title: WT, biological rep 2


title: FA19 WT 3

city: Atlanta

first: Gregory

last: Doho

state: GA

address: 1365B Clifton Rd NE

institute: Emory University

data_processing:
Illumina Casava1.7 software used for basecalling.
The raw reads were trimmed by removing adapter sequences and ambiguous nucleotides. Reads with quality scores less than 20 and length below 30 base pairs were all trimmed.
The resulting high-quality reads from each of the 3 replicate samples were mapped onto the N. gonorrhoeae FA1090 reference genome (GenBank accession: AE004969) using CLC Genomics Workbench software. For the reference mapping, at least 95% of the bases were required to align to the reference genome and a maximum of 2 mismatches were allowed.
The total number of reads mapped for each transcript was determined and then normalized to detect the reads per kilobase per million reads (RPKM) measure.
Genome_build: AE004969
Supplementary_files_format_and_content: EO_RNAseq_RPKM.xls contains RPKM values of all genes for six samples.

organism: Neisseria gonorrhoeae FA19

source_name: planktonic late-log phase cells

growth_protocol: Strains were grown in 50mL GCB broth (Difco) supplemented with Kellog’s supplements I and II (Dillard, 2001 Curr Protoc Micribiol) and 0.043% (v/v) sodium bicarbonate in a 37°C water bath with shaking at 200 rpm to late-log phase (OD600 = 0.600).

extract_protocol:
Total RNA was extracted from 1 mL culture samples using the Ambion RiboPure Bacteria kit according to manufacturer’s instructions. DNA was removed using Ambion TURBO DNA-free kit according to manufacturer’s instructions. rRNA was depleted by two treatments with Ambion MICROBExpress according to manufacturer’s instructions.
cDNA libraries were prepared using the Invitrogen SuperScript Double-stranded DNA synthesis kit and random hexameric primers according to manufacturer's instructions. cDNA librariers were amplified by 15 cycles of PCR to add adaptors with TruSeq indexes using the TruSeq Sample Preparation kit according to manufacturer's instructions.

value: wild-type


title: FA19mtr120 2

value: overexpresses mtrCDE


title: FA19 WT 1


title: FA19mtr120 3


title: FA19 WT 2


title: WT, biological rep 1


title: Jurkat Cells 236

status: Public on May 17 2013

city: Madison

first: Gloria

last: Sheynkman

state: WI

address: 1101 University Ave.

department: Chemistry

institute: UW-Madison

data_processing:
Basecalls performed using CASAVA version 1.8.
Annotated and unannotated junctions were detected using the Bowtie (v0.12.7) and Tophat (v1.4.0) splice-junction discovery programs (49, 50).  All default Bowtie parameters were used.  In Tophat, the mate inner distance was set to 150.  Two rounds of  Bowtie-Tophat processing were conducted with a supplied set of RefSeq gene model annotations in GTF format (7): the first round detected junctions only matching the gene annotation file (option --no-novel-junctions) and the second round detected all junctions, both aligning to the GTF file and novel (option -G).
RNA-Seq reads were processed by RSEM v1.1.20 (RNA-Seq by Expectation-Maximization) to estimate transcript abundances.  All default parameters used for both Bowtie (v0.12.7) and RSEM.
Genome_build: hg19

source_name: immortalized T-cell line

growth_protocol: The Jurkat cell line (TIB-152) was obtained from the American Type Culture Collection (ATCC, Manassas, VA).  Jurkat cell culture was grown in 10% Fetal Bovine Serum and 90% RPMI-1640 buffer (ATCC, Manassas, VA) at 37°C.  Cell concentration was measured using the TC10 Automated Cell Counter system (BioRad, Hercules, CA), which was validated via hemocytometer counting.  Before harvesting, cells were grown to ~1.3x106 cells/mL and had 95%+ viability as measured with the trypan blue assay.

extract_protocol:
~2e6 cells were pelleted, washed twice with cold PBS, and total RNA was extracted using the Trizol protocol.
RNA-Seq paired end libraries were prepared using the Illumina TruSeq RNA Sample Prep Rev. A (kit lot #6849988, Illumina, San Diego, CA).


title: Jurkat Cells 245


title: CD8+_Input_Biotin_ChIPseq

status: Public on May 16 2013

first: Willey

last: Liao

department: Bioinformatics

data_processing:
Basecalls performed using CASAVA v1.4
Bowtie v0.12.7 was used to identify sequences mapping uniquely to the genome
Peak calls were determined using BCP (http://rulai.cshl.edu/BCP)
Overlapping peaks between antibody and biotin ChIP methods were merged
Genome_build: mm9
Supplementary_files_format_and_content: BED files for uniquely mapping reads and called MACS peaks for Antibody, Biotin, and overlapping peaks are provided

source_name: CD8+ cells, Foxo1tagBirA mice

extract_protocol:
phenol-chloroform
Lysates were clarified from sonicated nuclei and Foxo1-DNA complexes were isolated with antibody or Streptavidin.  Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a kit from Epicentre. The blunt, phosphorylated ends were treated with Taq DNA polymerase and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15-19 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

value: CD8+, Foxo1tagBirA strepdavidin Input

tag: ChIP


title: CD8+_Input_Ab_ChIPseq

source_name: CD8+ cells, wild-type

value: CD8+, Foxo1 Ab Input


title: Jurkat Cells 216


title: CD8+_Foxo1_Biotin_ChIPseq

value: CD8+, Foxo1tagBirA strepdavidin ChIP

value: Strepdavidin


title: CD8+_Foxo1_Ab_ChIPseq

value: CD8+, Foxo1 Ab, ChIP

value: Foxo1 antibody (Abcam, Ab39670)


title: WT, biological rep1 (HITS-CLIP)

description: Protein-RNA complexes


title: BALBmockD1

city: Seattle

first: Wonjong

last: Moon

state: WA

address: 307 Westlake Avenue North, Suite 500

institute: Seattle BioMed

description: BALB/cJ-mock Day1

data_processing:
Illumina Genome Analyzer II GA2 HiSeq and CASAVA 1.8.2 for base calling
Additional quality control was applied to the datasets by clipping or removing poor quality reads and N's by ea-utils toolkit (http://code.google.com/p/ea-utils/), with -p 10 and -q 7 settings, then mapped to P. yoelii YM strain reference  genome using tophat version 1.4.1 with default parameters
The differential transcript abundances between samples were measured in Fragments per kilobase of transcript per million mapped fragments (FPKM) using  Cufflinks and Cuffdiff by a protocol from Trapnell C. et al. (2012) Nature.
RNA-Seq differential analysis was visualized by CummeRbund
Transcripts with P values of less than 0.01 and at least 4-fold difference in expression (i.e., log fold > 2) were selected to identify the most differentially regulated genes.
Coverage plots were generated to examine the RNA distribution of reads across selected genes-of-interest by comparing the number of unique reads present at each nucleotide position
Genome_build: Mus musculus UCSC version mm10
Supplementary_files_format_and_content: tab-delimited text files (cuffdiff gene_exp.diff) include gene differential FPKM values for each Sample

source_name: perfused mouse liver

extract_protocol:
RNA was isolated by the Qiagen RNeasy Miniprep Kit with an added DNaseI treatment module.
Library was constructed by Expression Analysis, Inc using the Illumina TruSeq RNA sample preparation kit using standard methods.

treatment_protocol: Mice were infected intravenously with 100,000 transgenic P. yoelii fabb/f- salivary gland sporozoites or with debris from uninfected mosquito salivary glands. One day post infection mouse livers were perfused with HBSS.  Livers were then flash frozen in liquid nitrogen until extraction.

value: BALB/cJ

value: mouse liver

tag: infection

value: 24 hours post mock infection with debris from mosquito salivary glands


title: C57BL6fabD1

description: C57BL/6-fabb/f- Day1

value: 24 hours post infection with Py fabb/f-knock out transgenic  P. yoelii sporozoite infection


title: C57BL6mockD1

description: C57BL6-mock Day1


title: WildtypeMouseRXRaInput

status: Public on May 15 2013

city: Sacramento

first: Yuqi

last: He

address: Rm3400, Research III, UC Davis Medical Center, 4645 2nd Ave

institute: UC Davis Medical Center

data_processing:
Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie
Peaks were called by using MACS
Genome_build: mm9
Supplementary_files_format_and_content: Bar file was generated from the bed file outputted from MACS

source_name: Liver

extract_protocol:
Grand in liquid nitrogen, then fixed, sonication, pull down by antibody
Illumina True-Seq Kit

value: Male

value: 3 Month

value: liver


title: FA19mtr120 1


title: MCIp_MO

city: Regensburg

first: Michael

last: Rehli

address: F.-J.-Strauss-Allee 11

department: Internal Med III

institute: University Hospital Regensburg

description: Methyl-CpG-immunoprecipitation

data_processing:
Sequence tags were mapped to the human reference sequence (GRCh27/hg19) using Bowtie1 and only uniquely mapped tags were used for downstream analyses. Parameters:  -q --best -m 1
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph file produced using the makeUCSCfile program in the HOMER software suite (http://biowhat.ucsd.edu/homer/)

source_name: Blood monocytes

growth_protocol: Peripheral blood monocytes were separated by leukapheresis of healthy donors, followed by density gradient centrifugation over Ficoll/Hypaque and subsequent counter current centrifugal elutriation in a J6M-E centrifuge. Monocytes were > 85% pure as determined by morphology and expression of CD14 antigen. To generate macrophages, isolated monocytes were cultured in endotoxin-free RPMI 1640 medium supplemented with vitamins, antibiotics, pyruvate, nonessential amino acids, 5 x 10-8 M β-mercaptoethanol, 2% human pooled AB-group serum on teflon foils for up to 7 days.

extract_protocol:
Chromatin Immunoprecipitation (Pham et al. 2012, Blood), Methyl-CpG-immunoprecipitation (Gebhard et al. 2006, Cancer Research)
DNA from chromatin immunoprecipitation (10-50ng) was adapter-ligated and PCR amplified using Illumina kit Preparing samples for ChIP sequencing of DNA (IP-102-1001) following the manufacturer’s protocol. ChIP fragments were sequenced for 36 cycles on Illumina Genome Analyzers I or II according to the manufacturer’s instructions. DNA from methyl-CpG-immunoprecipitation (10 ng) was treated like ChIP-derived DNA except that the amplification of the library was performed in the presence of 1M betain.

tag: chip antibody

value: MBD-Fc

value: Blood monocytes

tag: donor

value: B


title: miRNA from CMS (degradome)

city: Hangzhou

first: Jinghua

last: Yang

address: Yuhangtang 388

institute: Zhejiang University

description:
miRNA from CMS
degradome sequencing

data_processing:
Library strategy: RNA-Seq (degradome)
The small RNAs (sRNA) libraries of reproductive development from Brassica juncea CMS and MF were constructed by RNAs from all floral buds of one intact inflorescence. Deep sequencing generated a total number of 11845753 from MF and 3339182 from CMS raw reads (Supporting information Table S1). After removal of corrupted adapter sequences, reads with length <15 and >32 and junk reads, 7331575 and 3192676 mappable reads were finally obtained for MF and CMS libraries, respectively (Supporting information Table S1, S2). The majority of sRNA were 21-24 nucleotides (nt) for both two libraries that are within the typical size range for Dicer-derived products, in which the 21 nt sRNA was the most abundantly presented, followed by the 24 nt sRNA as the second largest percentage representing the class of endogenous small RNAs family (Fig. 1). We further compared the unique sRNAs between MF and CMS, which were 228 unique sRNAs for MF and 173 sRNAs for CMS. The number of the 21-24 nt unique sRNAs in MF was 173 and 141 in CMS, of which 21 nt unique sRNA was 98 in MF and 90 in CMS (Supporting information Table S3)
To identify the miRNAs from the reproductive development of Brassica juncea, sRNA sequences identified from Brassica juncea by deep sequencing were compared with the currently known mature plant miRNAs in miRBase 18.0. In miRbase, there are 291 Arabidopsis thaliana pre-microRNAs (pre-miRNAs) been reported corresponding to 328 mature miRNAs. Here, 99 pre-miRNAs corresponding to 94 miRNAs had been detected, besides, 63 5’ or 3’ new mature microRNAs corresponding to reported pre-miRNA in miRbase had been first time detected (supporting information Table S4, Table S5). There were 4 novel pre-miRNAs corresponding to 6 mature miRNAs originating from other pre-miRNAs except Arabidopsis thaliana. Among them, 3 5’ or 3’ new mature miRNAs corresponding to reported pre-miRNA in miRbase had been first time detected in Brassica juncea. The other 2 pre-miRNAs corresponding to 2 mature miRNAs come from 2 other plant species (Glycin max and Rehmannia glutinosa) can also been mapped to Arabidopsis thaliana genome had been first time detected here (Supporting information Table S6). 5 novel miRNAs originating from 7 pre-miRNAs that can not be mapped to Arabidopsis thaliana genome had been identified, of which these novel miRNAs were mapped to the other plant species genome and the extended sequences at the mapped positions of the genome potentially from hairpins (Supporting information Table S7). 17 novel miRNAs originating from pre-miRNAs that can not be mapped to Arabidopsis thaliana genome had been identified, of which these novel miRNAs were mapped to the other plant species genome and the extended sequences at the mapped positions of the genome, however failed in hairpin structure prediction (Supporting information Table S8). 13 other novel miRNAs which were conserved in plant species were observed, but they can not be found in Arabidopsis thaliana genome (Supporting information Table S9).
To identify new miRNAs, we removed all reads with low abundance (<5) from the sequence data. The data were used to query the non-coding RNAs sequence at database (ftp://ftp.arabidopsis.org/Sequences/ATH cDNA EST sequences FASTA/), the Rfam (ftp://ftp.sanger.ac.uk/pub/databases/Rfam/9.1/) database and repeat-repbase (http://www.girinst.org/repbase/update/index.html). Then, the consensus surrounding the genomic regions of each miRNA was retrieved and its secondary structure was formed according to public method (Zuker, 2003). All genomic loci-generating sRNAs that can be fold into the secondary structure were considered as miRNA candidate. Totally, 78 pre-miRNAs corresponding to 93 unique mature miRNAs had been first identified here, of which these candidate miRNAs were originated from predicted RNA hairpins (Supporting information Table S10). The secondary hairpin structures of the representative miRNAs were shown in Fig.2 and all the secondary hairpin structures of candidate miRNAs were last listed in Supporting Information Fig. S1. Interestingly, some new candidate miRNAs were organelle-derived non-coding sRNA, of which miRNAs, PC-5p-13, PC-3p-14, PC-3p-39, PC-5p-40, PC-3p-54, PC-3p-72, PC-5p-75, PC-5p-88 and PC-3p-90, are derived from chloroplast, and miRNAs, PC-5p-17, PC-3p-18, PC-3p-25 and PC-5p-26, are derived from mitochondria. Several pre-miRNAs sequences of new candidate miRNAs were aligned to be located in nuclear and mitochondrial genome, including pre-miRNA22/ pre-miRNA23, pre-miRNA24/ pre-miRNA25, pre-miRNA73/ pre-miRNA74 and pre-miRNA75/ pre-miRNA76 (Supporting information Table S10).  Finally, 290 miRNAs including known and new candidate were identified in reproductive development of MF and CMS Brassica juncea (Table 1).
miRNAs in MF and CMS were for differentially expression analysis using online service IDEG6 (http://telethon.bio.unipd.it/bioinfo/IDEG6_form/). The selection methods of differentially expression were Audic and Claverie, Fisher exact test and Chi-squared 2×2, and the selection threshold was 0.01 (http://telethon.bio.unipd.it/bioinfo/IDEG6_form/detail.html#AC). Finally, 47 miRNAs had been observed to be differentially expression between MF and CMS. Among these differentially expressed miRNAs, 25 miRNAs and 12 miRNAs were observed to be down- and up-regulated respectively according the sequencing reads (Table 2). The expressions of 8 selected miRNAs were tested by using quantitative real-time RT-PCR (qRT-PCR) analysis, which further validated the differentially expression data obtained from sequencing on the whole (Fig. 3). The expression pattern of 6 of 8 miRNAs was consistent with the sequencing reads. The expressions of ath-miR396b and ath-miR159_R-2 were shown no significant differences between CMS and MF, however, the expressions of these two miRNAs were detected to be down-regulated in CMS (Fig. 3).
we performed a genome-wide sequencing of miRNA-cleaved mRNA and Cleavland analysis based on recently developed high-throughput degradome sequencing technology (Addo-Quaye et al., 2008; German et al., 2008; Addo-Quaye et al., 2009).  The genome build(s)/miRBase version(s) used in the analysis of our data are TAIR10 of Arabidopsis arabidopsis genome from http://www.arabidopsis.org/ and miRbase version 19.0.

organism: Brassica juncea

source_name: floral buds

growth_protocol: when CMS and MF lines were flowering under long-day length.

extract_protocol:
Total RNA of four samples was extracted using mirVana miRNA Isolation Kit (Ambion, Austin, USA) according to the manufacturer's protocol. Briefly, 10 μg of RNA sample was size fractionated with YM-100 column (Millipore). The larger RNA fraction (>100 KDa) was removed. The smaller RNA fraction (<100KDa) was further fractioned on a 15% tris-borate-EDTA (TBE) urea polyacrylamide gel and 15-50nt fraction was obtained.
Subsequently, 5' and 3' RNA adapters were ligated to the small RNA by T4 RNA ligase (Promega), followed by reverse transcription and PCR products purification. Purified products were quantified on the TBS-380 mini-fluorometer (Turner Biosystems) using Picogreen dsDNA quantitation reagent (Invitrogen) and diluted to 10 nM and transferred for sequencing on the Illumina/Solexa G1 sequence (Illumina, San Diego, USA ).

tag: developmental stage

value: reproductive development

value: floral buds

tag: sequencing method

value: degradome sequencing

tag: line

value: cytoplasmic male sterile (CMS)


title: miRNA from MF (degradome)

description:
miRNA from MF
degradome sequencing

value: maintainer fertile (MF)


title: Input3

city: Heidelberg

first: Georg

last: Stoecklin

address: Im Neuenheimer Feld 280

institute: German Cancer Research Center

description: Sample 9

data_processing:
Basecalling with CASAVA v1.8.2
Mapping to the mouse genome NCBI build 37 with BWA.
Reads were summarized for gene models with the Bioconductor package easyRNASeq v1.2.5. The exon coordinates of RefSeq annotated mRNAs were obtained from the refGene table of the UCSC genome browser.
Differential abundance was tested for with the Bioconductor package DESeq v1.8.3.
Genome_build: NCBI build 37
Supplementary_files_format_and_content: The 'RoquinIP_processed.txt' file contains, for each sample, the count table obtained with easyRNASeq, the fold changes and the p-values computed with DESeq.

growth_protocol: RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (PAA Laboratories), 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in 5% CO2.

extract_protocol:
Cells were mechanically disrupted using a tissue lyser. RNA was isolated before (Input) or after IP (ctrl IP or Roquin IP) with TriFast reagent.
RNA libraries were prepared for sequencing using the TruSeq RNA Sample Preparation Kit.

treatment_protocol: RAW264.7 cells were treated with LPS (100 ng/ml, Sigma, L2630, E. coli serotype O111:B4) for 2 h.

tag: ip antibody/beads information

tag: molecule subtype

value: RNA purified from lysate before IP


title: Input1

description: Sample 7


title: Roquin_IP1

description: Sample 1

value: rabbit polyclonal, Bethyl, 514A and 515A plus protein A/G beads

value: RNA purified from Roquin IP


title: Ctrl_IP1

description: Sample 4

value: protein A/G beads alone

value: RNA purified from control IP without antibody


title: Roquin_IP3

description: Sample 3


title: CTCF_MO

description: Chromatin IP against CTCF

value: CTCF

value: A


title: CTCF_MAC

description: Chromatin IP against CTCF

source_name: Monocyte-derived macrophage

value: Monocyte-derived macrophage


title: Ctrl_IP3

description: Sample 6