title: TAP-Tagged Pax3

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

country: Canada

institute: Ottawa Hospital Research Institute

description: Pulldown of FLAG and TAP tagged Pax3.

data_processing: Solexa read alignment and peak calling. Solexa reads (36 bp) were aligned to mouse genome (NCBI37) by Eland (Illumina) with up to two mismatches.

source_name: myoblasts with TAP tagged Pax3

growth_protocol: Muscle satellite cells were isolated from adult C57BL/6 mice and cultured on collagen-coated plates (Roche-Boehringer) in Ham’s complete medium (Ham’s F-10, 20% fetal calf serum, 1% penicillin/streptomycin, and 2.5ng/ml human recombinant bFGF.

extract_protocol: Satellite cell-derived myoblasts stably expressing a C-terminus TAP tagged fusion protein were cross-linked using 1% formaldehyde in 1x PBS for 10 minutes. A solution containing 0.125 M glycine in 1x PBS was used for quenching the cross-linked reaction for 5 minutes at room temperature by gentle horizontal shaking. Cells were harvested by scraping and the cell pellet was dissolved in ChIP lysis buffer (40 mM Tris-HCl, pH 8.0; 1% Triton-X100; 4 mM EDTA; 300 mM NaCl) containing protease inhibitors (Aprotinin, 1 µg/ml; Leupeptin, 5µg/ml, Pepstatin A 1 µg/ml; PMSF, 1mM). Chromatin was fragmented by sonication in a water bath Bioruptor at 4ºC to an average length of 200 base pairs. The lysate was spun at 14K RPM for 15 minutes in a refrigerated centrifuge and the supernatant was diluted 1:1 in ChIP dilution buffer (40 mM Tris-HCl, pH 8.0; 4 mM EDTA, pH 8.0) plus protease inhibitors to reduce the concentration of salt and detergents. The first immunoprecipitation was done using anti 3xFLAG antibody conjugated to agarose beads (Sigma Aldrich) using 20 milligram of cell lysate (protein) as input for 2 hours at 4ºC. Beads containing antigen/antibody complex were washed 3 times with 10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 0.1% Triton-X100 containing protease inhibitors. Protein complex was eluted from M2-conjugated agarose beads using proteolytic cleavage with Tobacco Etch Virus (TEV) protease (Invitrogen) together with competition with 3xFLAG peptide (Sigma Aldrich) at 4ºC overnight. Two additional rounds of elution with 3xFLAG peptides were done using 200 µg/ml of 3xFLAG peptide in TBS (50 mM Tris-HCl, pH 7.4; 150 mM NaCl). The eluted product was used as input for the second affinity pull-down using His-Select nickel beads (Sigma-Aldrich) for three hours at 4ºC following manufacture’s recommendations. Beads were washed three times with wash buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM Imidazole). Final DNA/protein complex was eluted by 400 mM imidazole at room temperature. DNA/protein complexes were reverse cross-linked and ChIP DNA was purified by phenol/chloroform extraction followed by ethanol precipitation.

treatment_protocol: Pax3- and Pax7-cTAP were engineered with the in-frame addition of a C-terminal TAP tag, composed of 6 histidine residues and a 3x FLAG tag separated by a Tobacco Etch Virus (TEV) cleavage sequence. Retrovirus was produced in 293FT cells by transient transfection of expression constructs using Calcium Phosphate or linear polyethylenimine (PEI). Virus-containing supernatant was used to infect low-passage primary myoblasts supplemented with Ham’s complete medium and 8µg/ml polybrene. Transgenic cells were obtained by antibiotic selection and maintained in Ham’s complete medium containing 1.0 µg/ml puromycin.

tag: strain

value: BALB/c

tag: tissue of origin

value: skeletal muscle

tag: cell type

value: primary myoblasts


title: TAP-Tagged Pax7

status: Public on May 22 2012

type: SRA

description: Pulldown of FLAG and TAP tagged Pax7.

library_source: genomic

molecule: genomic DNA

organism: Mus musculus

source_name: myoblasts with TAP tagged Pax7


title: Control

description: Control run with no tagged proteins.

source_name: myoblasts


title: Downy infected 24h

status: Public on May 21 2012

city: Legnaro

first: Alessandro

last: Vannozzi

state: PD

address: via dell'Università 16

department: DAFNAE

country: Italy

institute: University of Padova

library_source: transcriptomic

data_processing:
The evaluation of gene expression was performed with both CLC genomic workbench and ERANGE 3.1 softwares. The trascriptional activity of each gene was defined as the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Genome Build:
s_3_sequence rpkm.txt: 12X V1 PN40024

molecule: total RNA

organism: Vitis vinifera

source_name: V. vinifera leaf tissues

growth_protocol: Dormant Pinot noir cutting for glasshouse propagation were obtained from field grown vines at the "Lucio toniolo" experimental farm of the University of Padua.

extract_protocol: mRNA Sequencing Sample Preparation kit'. Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads, fragmented into small pieces using divalent cations under elevated temperature, cDNA was synthesized by reverse transcription and standard blunt-ending plus add ‘A’ was performed. Then, Illumina adapters were ligated to the ends of the cDNA fragments. After ligation reaction and separation of not ligated adapters, samples were amplified by PCR to selectively enrich those cDNA fragments in the library having adapter molecules at both ends.

treatment_protocol: Leaf discs of 15 mm of diameter were punched from healty leaves detached from glasshouse grown V. vinifera cv. Pinot noir vines. Discs were obtained from leaves belonging to different plants and showing similar age based o size and node position in plants, treated with different biotic and abiotic stresses and incubated upside down on 3MM moist filter paper in large Petri dishes untill harvest at which point discs were immediately frozen in liquid itrogen and stored at -80°C. The punching of discs was considered as a wounding treatment. For the UV-C treatment, discs were irradiated at 254nm (0.36 J/cm3) for 10 minutes at a distance of 10 cm from the UV-C source. Downy mildew infection was carried out spraying a solution containing downly mildew sporangia at a concentration of 105 sporrangia ml-1. Pinot noir leaf discs were sampled at 0, 24 and 48 h after each treatment and total RNA extracted. The 0h timepoint was considered common for the three treatments.

tag: tissue

value: leaf discs

tag: infection

value: P. viticola

tag: time

tag: cultivar

value: Pinot noir


title: UV-C treated 48h

data_processing:
The evaluation of gene expression was performed with both CLC genomic workbench and ERANGE 3.1 softwares. The trascriptional activity of each gene was defined as the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Genome Build:
s_7_sequence rpkm.txt: 12X V1 PN40024

tag: treatment

value: UV-C


title: UV-C treated 24h

data_processing:
The evaluation of gene expression was performed with both CLC genomic workbench and ERANGE 3.1 softwares. The trascriptional activity of each gene was defined as the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Genome Build:
s_6_sequence rpkm.txt: 12X V1 PN40024


title: Wounded 48h

data_processing:
The evaluation of gene expression was performed with both CLC genomic workbench and ERANGE 3.1 softwares. The trascriptional activity of each gene was defined as the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Genome Build:
s_2_sequence rpkm.txt: 12X V1 PN40024

value: Wounding


title: Wounded 24h

data_processing:
The evaluation of gene expression was performed with both CLC genomic workbench and ERANGE 3.1 softwares. The trascriptional activity of each gene was defined as the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Genome Build:
s_1_sequence rpkm.txt: 12X V1 PN40024


title: Control

data_processing:
The evaluation of gene expression was performed with both CLC genomic workbench and ERANGE 3.1 softwares. The trascriptional activity of each gene was defined as the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Genome Build:
s_8_sequence rpkm.txt: 12X V1 PN40024

value: Control


title: Stanford_ChipSeq_HUVEC_c-Jun_std

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

country: USA

institute: ENCODE DCC

organism: Homo sapiens

source_name: HUVEC

extract_protocol: Instrument model unknown. ("Illumina Genome Analyzer" specified by default). For more information, see http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeSydhTfbs

tag: lab

value: Stanford

tag: lab description

value: Snyder - Stanford University

tag: datatype

value: ChipSeq

tag: datatype description

value: Chromatin IP Sequencing

tag: cell

value: HUVEC

tag: cell organism

value: human

tag: cell description

value: umbilical vein endothelial cells

tag: cell karyotype

value: normal

tag: cell lineage

value: mesoderm

tag: cell sex

value: U

tag: treatment description

value: No special treatment or protocol applies

tag: antibody

value: c-Jun

tag: antibody antibodydescription

value: Rabbit polyclonal to amino acids 1-79 mapping at the N-terminus of c-Jun p39 of human origin. Antibody Target:c-Jun

tag: antibody targetdescription

value: Heterodimer of Fos and Jun constitute transcription factor AP1.  Proto-oncogene c-Jun is a leucine-zipper.

tag: antibody vendorname

value: Santa Cruz Biotech

tag: antibody vendorid

tag: control

value: std

tag: control description

value: Standard input signal for most experiments.

tag: controlid

tag: replicate


title: SyntheticPromotersExpressionProfiling rep 2

status: Public on May 20 2012

city: Rehovot

first: Eilon

last: Sharon

address: 234 Herzl Street

department: Computer Science And Applied Mathematics

country: Israel

institute: Weizmann Institute of Science

data_processing:
Sequencing reads were mapped to a reference sequence set that contained all combinations of strain and expression bin barcode sequences using SHRiMP. This provides the fraction of cells with each promoter in each expression bin.
Mean expression of each expression bin was extracted from the FACS measurements.
Mean expression for each promoter was computed using a weighted mean of the expression bins mean expression values according to the promoter strain distribution across the bins.
Genome_build: Synthetic promoters
Supplementary_files_format_and_content: PromoterSequenceAndExperimentsResults.xlsx. Tabular file that contains the following columns: 1.LibraryID – unique index. 2.Description – description of the elements which compose each synthetic promoter. 3.Sequence – synthetic promoter variable region sequence. 4.NumberOfReads Replicate 1 – Number of reads obtained for each promoter strain. Estimation of expression mean may improve with larger number of reads. 5.NumberOfReads Replicate 2 – Number of reads obtained for each promoter strain. Estimation of expression mean may improve with larger number of reads. 6.Expression Replicate1 – Mean expression induced by the synthetic promoter. 7.Expression Replicate2 – Mean expression induced by the synthetic promoter.

organism: Saccharomyces cerevisiae

source_name: Exponentially growing yeast cells

growth_protocol: To adapt cells to the medium of the sorting, stationary yeast library cells were diluted in SC-Gal-URA synthetic complete media with 2% galactose and without uracil) medium without amino acids, except for histidine and leucine, and were grown to the stationary phase. Next, this culture was diluted again in similar medium (to OD600 0.03–0.05) and grown to the mid-exponential phase (OD600 0.5–1.5) for sorting.

extract_protocol: Cells were sorted to expression bins with the FACSAria cell sorter (Beckton-Dickinson) according to their YFP reporter gene expression level. The library was sorted using two strategies. In one strategy (replicate 1), the cells were sorted three times recursively into four bins, producing a total of 64 bins (we first sorted all the cells into four bins, applied the same sorting procedure to the cells in each bin and then applied this procedure again to the cells of each bin sorted at this second level). In another strategy (replicate 2), we sorted the cells directly into 16 bins. In each sorting strategy, the expression bins contained equal fractions of the library cells, and we collected a total of 8,000,000 cells. Sorted cells were grown in 5 ml SCD-URA medium to the stationary phase. One million cells from each bin were taken for colony PCR using specific primers corresponding to the promoter region of the plasmid. The sample PCR product was sequenced using Applied Biosystem’s SOLiD high-throughput sequencing platform (AB SOLiD).

tag: strain

tag: medium

value: SC-Gal-URA (synthetic complete media with 2% galactose and without uracil)


title: SyntheticPromotersExpressionProfiling rep 1


title: spleen

status: Public on May 18 2012

city: shenzhen

first: zhiyong

last: huang

state: Guangdong

address: F5, Main Building, Beishan Industrial Zone, Beishan Road, Yantian District

country: China

institute: BGI-Shenzhen, Shenzhen 518083, China

description: Sequenced reads were generated from spleen in yak

data_processing: use Bos grunniens v2 genome as reference, sequenced reads were aligned onto the corresponding reference using TopHat v1.0.12 with default parameters except  -r 20 --mate-std-dev 5 -g 20 -I 10000 --solexa1.3-quals

organism: Bos grunniens

source_name: spleen

extract_protocol:
total RNA is extracted by TRIzol Reagent
For RNA-seq, poly-A+ RNA was isolated with 30 oligo-dT-coupled beads from 20 μg total RNA of each sample. First strand cDNA synthesis was performed with random hexamers and Superscript II reverse transcriptase (Invitrogen). The second strand was synthesized with E. coli DNA PolI (Invitrogen). Double stranded cDNA was purified with Qiaquick PCR purification kit (Qiagen), and sheared with a nebulizer (Invitrogen) to 100-500 bp fragments. After end repair and addition of a 3’ dA overhang the cDNA was ligated to Illumina PE adapter oligo mix, and size selected to 200±20 bp fragments by gel purification. After 15 cycles of PCR amplification the libraries were sequenced using a 1G Illumina HiSeq 2000 and the paired-end sequencing module.

tag: tissue

value: spleen


title: lung

description: Sequenced reads were generated from lung in yak

source_name: lung

value: lung


title: stomach

description: Sequenced reads were generated from stomach in yak

source_name: stomach

value: stomach


title: heart

description: Sequenced reads were generated from heart in yak

source_name: heart

value: heart


title: liver

description: Sequenced reads were generated from liver in yak

source_name: liver

value: liver


title: brain

description: Sequenced reads were generated from brain in yak

source_name: brain

value: brain


title: Downy infected 48h

data_processing:
The evaluation of gene expression was performed with both CLC genomic workbench and ERANGE 3.1 softwares. The trascriptional activity of each gene was defined as the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Genome Build:
s_4_sequence rpkm.txt: 12X V1 PN40024

value: leaf discs

tag: infection

value: P. viticola

tag: time

tag: cultivar

value: Pinot noir


title: Dictyostelium cc cold synchronized t4

city: Montreal

first: Kimchi

middle: Audrey

last: Strasser

state: Quebec

address: 7141 Sherbrooke Street West

department: Biology

institute: Concordia University

description: polyA RNA

data_processing: Reads from barcoded samples were split into separate files according to their barcode, and the barcode sequences were trimmed off. Reads were aligned with BowTie v0.12.7 to the Dictyostelium discoideum chromosomal DNA sequences downloaded from dictybase.org on August 12, 2010, augmented with splice junction sequences. Introns were first located by mapping the pooled reads to the genomic sequence with BLAT; reads that aligned with >95% identity and coverage, but with gaps, were examined for the presence of plausible introns in the gaps. Splice junction sequences were constructed by concatenating 41 bases of sequence from the two flanks of the introns thus identified. Read alignments to the splice junction sequences were translated into gapped alignments to the genome. Reads that mapped equally well to more than one location were randomly assigned to one of the locations with probability proportional to the local depth of coverage by uniquely-mapped reads.

organism: Dictyostelium discoideum

source_name: vegetative cells

growth_protocol: Vegetative cells were grown in HL5 medium and harvested at a density of 2E6 cells/ml. For development, cells were dispersed on Whatman 50 filters overlaid on Millipore absorbent pads moistened with LPS. Aggregates were harvested in the early culminant stage. Cells were synchronized in the cell cycle using the cold shock protocol (Maeda (1986) J Gen Microbiol 132, 1189-1196).

extract_protocol: Libraries were prepared according to Illumina's instructions accompanying the mRNA-Seq Sample Prep Kit.

tag: background/strain

tag: genotype

value: wild type

tag: developmental stage

value: vegetative

tag: treatment

value: cell cycle cold synchronized

tag: time (hr)


title: Dictyostelium cc cold synchronized t12

description: polyA RNA


title: Dictyostelium cc cold synchronized t8

description: polyA RNA


title: Dictyostelium cc cold synchronized t10

description: polyA RNA


title: Dictyostelium cc cold synchronized t9

description: polyA RNA


title: Dictyostelium cc cold synchronized t7

description: polyA RNA


title: Dictyostelium cc cold synch t2, rep2

description: polyA RNA


title: Dictyostelium cc cold synchronized t5

description: polyA RNA


title: Dictyostelium cc cold synchronized t13

description: polyA RNA


title: Dictyostelium cc cold synchronized t11

description: polyA RNA


title: Dictyostelium cc cold synchronized t3

description: polyA RNA


title: Dictyostelium cc cold synch t1, rep2

description: polyA RNA


title: Dictyostelium cc cold synch t1, rep1

description: polyA RNA


title: Dictyostelium cc cold synch t1, rep3

description: polyA RNA


title: Dictyostelium cc cold synch t0 rep2

description: polyA RNA


title: Dictyostelium rblA disruptant early culminant

description:
4-rblA KO early culm
polyA RNA

source_name: developing cells

value: rblA disruptant clone3A6-12

value: early culminant


title: Dictyostelium cc cold synch t0 rep1

description: polyA RNA


title: Dictyostelium cc cold synch t2, rep1

description: polyA RNA


title: Dictyostelium rblA disruptant vegetative

description:
2-rblA KO veg
polyA RNA


title: Dictyostelium wt vegetative

description:
1-Ax2 veg
polyA RNA


title: Dictyostelium cc cold synchronized t6

description: polyA RNA


title: H3K9me3_ChIPSeq

status: Public on May 17 2012

city: Milan

first: Francesca

middle: R.

last: Santoni de Sio

address: via Olgettina 58

department: SR-TIGET

institute: San Raffaele Scientific Institute

data_processing:
Basecalls performed using GAP Pipeline version 1.7
Reads were aligned to the Mus musculus genome (assembly NCBI37/mm9) using bowtie.
Reads with more than four matches were excluded.
peak calling was performed by using the ChIP-Seq Analysis Server (http://ccg.vital-it.ch/chipseq/)
Genome_build: mm9
Supplementary_files_format_and_content: bed files produced by the Chip-seq on line analysis tool (http://ccg.vital-it.ch/chipseq/)

source_name: B cell enriched splenocytes

growth_protocol: Freshly isolated cells

extract_protocol: After magnetic separation, 10^7 unbound cells/sample were crosslinked by 1% formaldehyde and sonicated by Branson sonicator. Chromatin was immunoprecipitated by using an affinity-purified rabbit polyclonal antibody raised against KAP1 amino acids 20–418 (RBCC) (Schultz DC Gen Dev 2001, kindly provided by Dr. Rauscher) or polyclonal H3K9me3 antibody (Diagenode). Detailed protocol for Chromatin immunoprecipitation is described elsewhere (Meylan S. et al. BMC Genomics 2011). After de-crosslinking of both IP and total input, DNA was quantified with the Qubit fluorometer (Invitrogen) and 10 ng were used to prepare libraries for sequencing, following the ChIP Seq library preparation protocol (including End Repair for blunted end fragments, addition of “A” base to the 3’ end and ligation of adapters to DNA fragments (kit IP-102-1001). Ligation products were then purified on 2% E Gels Size Select (Invitrogen, fragments of 200 bp recovered), followed by enrichment of DNA fragments by PCR (18 cycles). No more than two libraries were loaded per gel to avoid cross contamination. Libraries were purified on AMP XP beads (Agencourt), quantified with Qubit fluorometer and size distribution determined on the Agilent Bioanalyzer (High Sensitivity DNA chip).

treatment_protocol: B cells were enriched by negative selection. Total splenocytes were treated with Ammonium Chloride solution to lyse red cells and magnetic negative selection was performed by staining with biotin-conjugated anti-CD11b and anti-CD3 antibodies copled with biotin-binder magnetic beads (Invitrogen).

tag: cell type

value: B cell enriched splenocytes

tag: chip antibody

value: H3K9me3 (Diagenode, pAb-056-050)

tag: genotype/variation

value: wildtype


title: input DNA, 1


title: KAP1_KO_ChIPSeq

source_name: B cell enriched splenocytes from KAP1 KO mouse

value: RBCC (Schultz et al. Genes Dev. 2001)

value: KAP1 KO


title: KAP1_ChIPSeq


title: input DNA, 2


title: H3K9me3_KO_ChIPSeq


title: H1 human ES cells (paired-end)

city: Dallas

first: Gary

middle: Chung

last: Hon

state: TX

address: 5323 Harry Hines Blvd.

department: OB/GYN

institute: UT Southwestern

description: 5-hmC whole genome bisulfite sequencing

data_processing: Reads were processed as previously reported(Hon et al., 2012; Lister et al., 2009). Briefly, raw reads were trimmed for low quality bases and adapter sequences. Then, cytosine bases were computationally replaced with thymines, mapped with the Bowtie program (Langmead et al., 2009) against computationally converted copies of hg18 or mm9, and mapped reads were resorted to their pre-computationally-converted bases. PCR duplicates were removed for each PCR amplification reaction using the Picard program (http://picard.sourceforge.net). To eliminate reads not bisulfite converted, reads having more than 3 base calls in non-CG context were removed, as previously (Lister et al., 2009). All libraries were then merged and indexed by the SAMtools suite (Li et al., 2009).

source_name: H1 human ES cells

growth_protocol: H1 human ES cells were cultured on matrigel plates in mTeSR1 feeder-independent maintenance medium (Stemcell Technologies Cat. No. 05850) supplemented with 1x pen/strep. The culture was passaged every 2-4 days.

extract_protocol: Purified genomic DNA was fragmented to an average size of 300bp with a Biorupter 300 (high power, 15s on, 15s off, 20 cycles). Spike in 5mC conversion controls were added prior to sonication at 0.5% of total genomic DNA. Glycosylation reactions were performed in a 50 ?l solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, 100 ng/?l sonicated humun or mouse genomic DNA, 200 ?M UDP-Glc, and 1 ?M wild-type ?GT. The reactions were incubated at 37 oC for 1 h. After the reaction, the DNA was purified by QIAquick Nucleotide Removal Kit (Qiagen). The oxidation reactions were performed in a 50 ?l solution containing 50 mM HEPES buffer (pH 8.0), 100 ?M ammonium iron (II) sulfate, 1 mM ?-ketoglutarate, 2 mM ascorbic acid, 2.5 mM DTT, 100 mM NaCl, 1.2 mM ATP, 10 ng/?l glycosylated DNA and 3 ?M recombinant mTet1. The reactions were incubated at 37 oC for 1.5 h. After proteinase K treatment, the DNA was purified with Micro Bio-Spin 30 Columns (Bio-Rad) first and then applied to QIAquick PCR Purification Kit (Qiagen). 500ng-1ug TABS treated DNA was end-repaired, adenylated, and ligated to methylated (5mC) adapters (Illumina) according to standard Illumina protocols for genomic DNA library construction, maintaining the proper molar ratios of adapter to insert. Adapter ligated fragments with 200-400bp inserts were gel purified by 2% agarose gel electrophoresis and sodium-bisulfite treated using the MethylCode kit (Invitrogen). Bisulfite treated adapter-ligated DNA was amplified by PCR with the following conditions in a 50uL final reaction volume:  2.5U PfuTurbo Cx Hotstart DNA polymerase, 5uL 10X PfuTurbo Cx reaction buffer, 0.75uL 20mM dNTPs, 5uL Illumina PCR Primers. Cycling parameters: 95ºC 5min, 98ºC 30 sec, followed by 7-9 cycles of 98ºC 15 sec, 60ºC 30 sec, 72ºC 4 min, and ending with 72ºC 10 min. The number of PCR cycles used was determined by quantification of bisulfite treated adapter-ligated DNA by qPCR (KAPABiosystems library quant kit for Illumina libraries) such that the final library concentration obtained was approximately 20nM. Final sequencing libraries were purified by 2% agarose gel electrophoresis and quantified by qPCR (KAPABiosystems library quant kit for Illumina libraries). Up to 3 separate PCR reactions were performed per sample.

treatment_protocol: Genomic DNA library coupled with protection of 5-hmC, Tet-assisted 5mC oxidation, and bisulfite treatment

tag: cell line


title: E14Tg2a

description: 5-hmC whole genome bisulfite sequencing

data_processing:
Reads were processed as previously reported(Hon et al., 2012; Lister et al., 2009). Briefly, raw reads were trimmed for low quality bases and adapter sequences. Then, cytosine bases were computationally replaced with thymines, mapped with the Bowtie program (Langmead et al., 2009) against computationally converted copies of hg18 or mm9, and mapped reads were resorted to their pre-computationally-converted bases. PCR duplicates were removed for each PCR amplification reaction using the Picard program (http://picard.sourceforge.net). To eliminate reads not bisulfite converted, reads having more than 3 base calls in non-CG context were removed, as previously (Lister et al., 2009). All libraries were then merged and indexed by the SAMtools suite (Li et al., 2009).
Genome Build:
mESC.mm9.+.bed.gz: mm9
mESC.mm9.-.bed.gz: mm9

source_name: mouse embryonic stem cells

growth_protocol: E14 (E14Tg2A) ES cell lines were cultured in feeder-free gelatin-coated plates in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen Cat. No. 11995) supplemented with 15 % FBS (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma), 1x non-essential amino acids (GIBCO), 1,000 units/ml LIF (Millipore Cat. No. ESG1107), 1x pen/strep (GIBCO). The culture was passaged every 2 days.

extract_protocol: Purified genomic DNA was fragmented to an average size of 300bp with a Covaris S2 (10% duty cycle, intensity 4, 200 cycles per burst, 80 sec). Spike in 5mC conversion controls were added prior to sonication at 0.5% of total genomic DNA.  Glycosylation reactions were performed in a 50 μl solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, 100 ng/μl sonicated humun or mouse genomic DNA, 200 μM UDP-Glc, and 1 μM wild-type βGT. The reactions were incubated at 37 oC for 1 h. After the reaction, the DNA was purified by QIAquick Nucleotide Removal Kit (Qiagen). The oxidation reactions were performed in a 50 μl solution containing 50 mM HEPES buffer (pH 8.0), 100 μM ammonium iron (II) sulfate, 1 mM α-ketoglutarate, 2 mM ascorbic acid, 2.5 mM DTT, 100 mM NaCl, 1.2 mM ATP, 10 ng/μl glycosylated DNA and 3 μM recombinant mTet1. The reactions were incubated at 37 oC for 1.5 h. After proteinase K treatment, the DNA was purified with Micro Bio-Spin 30 Columns (Bio-Rad) first and then applied to QIAquick PCR Purification Kit (Qiagen). 500ng-1ug TABS treated DNA was end-repaired, adenylated, and ligated to methylated (5mC) adapters (Illumina TruSeq Genomic DNA adapters) according to standard Illumina protocols for genomic DNA library construction, maintaining the proper molar ratios of adapter to insert. Adapter ligated fragments with 200-400bp inserts were gel purified by 2% agarose gel electrophoresis and sodium-bisulfite treated using the MethylCode kit (Invitrogen). Bisulfite treated adapter-ligated DNA was amplified by PCR with the following conditions in a 50uL final reaction volume:  2.5U PfuTurbo Cx Hotstart DNA polymerase, 5uL 10X PfuTurbo Cx reaction buffer, 1.25uL 10mM dNTPs, 5uL Illumina TruSeq PCR Primer Cocktail. Cycling parameters: 95ºC 5min, 98ºC 30 sec, followed by 7-9 cycles of 98ºC 10 sec, 65ºC 30 sec, 72ºC 30 sec, and ending with 72ºC 5 min. The number of PCR cycles used was determined by quantification of bisulfite treated adapter-ligated DNA by qPCR (KAPABiosystems library quant kit for Illumina libraries) such that the final library concentration obtained was approximately 20nM. Final sequencing libraries were purified with AMPure XP beads and quantified by qPCR (KAPABiosystems library quant kit for Illumina libraries). Up to 3 separate PCR reactions were performed per sample.


title: Dictyostelium Ax2 early culminant

description:
3-Ax2 early culm
polyA RNA


title: H1 human ES cells

description: 5-hmC whole genome bisulfite sequencing


title: HIV_24hr_rep2

status: Public on May 16 2012

city: Seattle

first: Michael

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

data_processing: We mapped the 75-nt reads to human ribosomal sequences using the short-read aligner software Bowtie to remove potential rRNA sequences. To quantify transcript expressions we mapped all reads that remained unmapped to the human reference genome (hg19, build GRCh37, downloaded from UCSC genome browser, http://genome.ucsc.edu) using the gapped aligner software TopHat. RefSeq transcript annotations were supplied to facilitate the mapping of reads spanning known splicing junctions. Based on these human genome mapping results we then estimated the expression abundances at both transcript and locus levels for RefSeq-annotated genes using the transcript assembly software Cufflinks. Read sequences that mapped to more than one genomic location were excluded from expression quantification. For visualization BAM files were generated using TopHat and samtools and displayed using the UCSC genome browser.

molecule: polyA RNA

source_name: polyA RNA from HIV-infected SUPT1 at 12 hours post-infection, replicate 3

extract_protocol: Total RNA was extracted from 5x106 cells per sample using mirVana kit (Applied Biosystems/Ambion, Austin, TX) and the quality and concentration were determined by Agilent 2100 Bioanalyzer. Samples were submitted for sequencing to Illumina FastTrack Sequencing Services (Hayward, CA). cDNA libraries were generated using Illumina mRNA-SEQ kit. The quality and concentration of these libraries were determined by Agilent 2100 Bioanalyzer. Libraries were clonally amplified on a cluster generation station using Illumina Version 4 cluster generation reagents to achieve a target density of approximately 300K/mm2 in a single channel of a flow cell.

value: CD4+ human T-cell line SupT1

value: HIV

value: 24 hours post-infection


title: Mock_24hr_rep1

source_name: polyA RNA from HIV-infected SUPT1 at 24 hours post-infection, replicate 2

value: Mock


title: Mock_12hr_rep1

source_name: polyA RNA from mock-infected SUPT1 at 24 hours post-infection, replicate 2

value: 12 hours post-infection


title: Mock_12hr_rep2

source_name: polyA RNA from mock-infected SUPT1 at 24 hours post-infection, replicate 3


title: 1 min (single end)

city: Evanston

first: Ji-Ping

last: Wang

address: 2006 Sheridan Road

institute: Northwestern University

description: genome build: SGD SAC2

data_processing: For the single-end samples including 20091030_20min, 20100111_1_5min, 20100111_1min and 20100111_20min, we used Software mapreads-2.4.1 for alignment allowing for up to 3 mismatches. For the paired end samples including PE_1_5min and PE_20min,we used Software BioScope-1.2.1 for alignment allowing for up to 3 mismatches for the F3 end and 2 mismatches for the F5 end. For all samples, only reads that were uniquely mapped to yeast genome of SGD SAC2 version were kept for the following analyses.The High-throughput sequencing  and the  related alignment were performed at the Northwestern University Genomics Core Facility.

source_name: Nucleosomal DNA

growth_protocol: The H4S47C S. cerevisiae strain was grown at 30 degrees Celcius in YPD (10% yeast extract, 20% peptone and 20% dextrose) to a density of ~1.5x107 cells/ml.

extract_protocol: DNA was purified by phenol-chloroform extraction and subsequent ethanol precipitation. The sample was RNased (1mg/ml final) overnight at 42oC and run on a 2% agarose gel.  The shortest DNA band, corresponding to the center-to-center distance of two nucleosome neighbors, was cut out of gel and the DNA was extracted.  The ends of the chemically mapped DNA were blunted using first NEB’s Klenow Polymerase large fragment (cat # M0210L) then processed further using Lucigen’s DNATerminator End Repair Kit (cat # 40035-2).  The sequence of events in the blunting and phosphorylation of the mapped DNA ends is crucial for the subsequent analysis of the mapped products. Initial treatment with Klenow Polymerase large fragment first removes 3’ overhands and fills in 5’ overhangs to create blunted DNA fragments. The use of a single polymerase, such as Klenow, creates a more homogenous set of DNA ends relative to the hydroxyl radical cleavage sites. Lucigen’s DNATerminator End Repair Kit was used primarily for its phosphorylation abilities. However any possible residual overhanging DNA ends would be blunted by this treatment (the kit blunts all single stranded DNA overhangs.  ABI SOLiD adapters were ligated to the blunted and phosphorylated DNA ends. Nick translation and PCR amplification were completed following ABI’s SOLiD Fragment Preparation protocol. All samples were sequenced with ABI’s SOLiD next generation sequencing system.

treatment_protocol: For a 500mL culture, cells were collected and quickly washed with 1M sorbitol and immediately resuspended in 5mL of permeabilizing buffer (1M sorbitol, 5mM BME and 0.5mg of lyticase Sigma cat# L5263-200KU) and incubated at room temperature with rotation for 5 minutes.   Permeabilized cells were washed 2x in 5ml of 1M sorbitol with 0.1% NP-40 and resuspended in 2mL of labeling buffer (1M sorbitol, 50mM NaCl, 10mM Tris-HCl pH 7.5, 5mM MgCl2, 0.5mM spermidine, 0.15mM sperimine, 0.1% NP-40). Immediate permeablization is necessary to deplete cellular ATP and minimize salts, reducing the capabilities of a nucleosome to shift positions. We chose not to formaldehyde cross-link because of the potential effects cross-linking could have on the spatial positioning of the cysteine and the label in relation to the DNA backbone. The thiol reactive label - N(1,10 phenanthroline- 5-yl) iodoacetamide  (Biotium cat# 92015), was dissolved in DMSO to a concentration of 7mM and added to the cells for a final concentration of 1.4mM of label and 20% total volume of DMSO.  This concentration of DMSO was chosen to facilitate entry of the thiol reactive label into the cells.  Research from several publications indicates that DMSO at this concentration does not affect protein-protein and protein-DNA interactions. Additionally, we are not concerned about DMSO induced DNA melting because of the low temperature used during the labeling process.   The light-sensitive labeling reaction was incubated at room temperature, with rotation for 2 hours, and then overnight at 4oC. This reaction was then washed 2x in 10mL of 1M sorbitol and 0.1% NP-40, then resuspended in 1.5mL mapping buffer (1M sorbitol, 2.5mM NaCl, 50mM Tris-HCl pH 7.5, 5mM MgCl2, 0.5mM spermidine, 0.15mM spermine and 0.1% NP-40).  Cupric chloride (CuCl2) was added to a final concentration of 150mM and incubated for 5min.  The cells were washed 2x in 10mL of mapping buffer and resuspended in 1.5mL mapping buffer. Beta-mercaptopropionic acid (MPA) was added to a final concentration of 6mM, and incubated for 5min, reducing the copper to cuprous chloride.  The chemical mapping reaction was then initiated by the addition of hydrogen peroxide to a final concentration of 6mM.  The reaction was mixed well and incubated for 20 minutes (or 1min or 1.5min - the reaction time did not affect nucleosomes map).  The mapping reaction was quenched by the addition of Neocuprine (Sigma), 2.8mM final.  Once quenched the cells were spun down and resuspended in 1mL of 1mM Tris-HCl pH 7.5 and 0.1mM EDTA.  To disrupt histone-DNA contacts, SDS and NaCl were added to a final concentration of 2% and 2M respectively and incubated for 1 hour.

value: H4S47C, ∆HHF2, BY4741 (MATa, his3∆1, leu2∆0, met15∆0, ura3∆0)

tag: growth phase

value: Mid-log phase yeast

tag: incubation time

value: 1 min

tag: single or paired-end

value: single


title: 20 min (single end) -2

description: genome build: SGD SAC2

tag: strain

value: 20 min


title: 20 min (paired end)

description: genome build: SGD SAC2

value: paired-end


title: 1.5 min (paired end)

description: genome build: SGD SAC2

value: 1.5 min


title: Mock_24hr_rep3

source_name: polyA RNA from HIV-infected SUPT1 at 24 hours post-infection, replicate 3


title: PlatypusFibroblastMale

city: Lausanne

first: Philippe

last: Julien

address: Batiment Genopode

department: CIG

country: Switzerland

institute: University of Lausanne

data_processing:
For fibroblast libraries: the sequencing files were processed as described in the Brawand, Soumillon, Necsulea et al publication in nature (PMID: 22012392). Briefly, the sequencing reads were mapped to the genome of the platypus (Ensembl annotation 57) using TopHat and Bowtie. A custom script was used to compute RPKM values resolving ambiguities of reads with multiple mapping positions (see original publication for detailed method). The output tab-separated file provides Ensembl IDs of platypus genes (Ensembl annotation 57) and the associated RPKM value (not log-transformed).
For the ovary library: the fastq file was aligned to the platypus genome (Genome and gene annotation: Ensembl 62) using the TopHat software (version 1.0.13) with the following arguments: -p 4 -a 8 -i 40 -I 1000000 -F 0 -m 1 --coverage-search --microexon-search --solexa1.3-quals --butterfly-search --library-type fr-secondstrand. The output (BAM files) was processed by Cufflinks (version 1.0.3) with the following parameters (-p 4 --library-type fr-secondstrand --multi-read-correct --upper-quartile-norm) in order to compute final gene expression levels estimates (FPKM). The provided processed data is presented as a standard cufflinks genes output (http://cufflinks.cbcb.umd.edu/manual.html#tracking_format).
Genome Build:
PlatypusFibroblastMale.txt: Ensembl 57

organism: Ornithorhynchus anatinus

source_name: Fibroblast

extract_protocol: Total RNA was extracted using the Trizol (Invitrogen) procedure or RNAeasy/RNAeasy Lipid/miRNeasy (Qiagen) column purification kits as indicated. RNA quality was assessed using an Agilent 2100 Bioanalyzer. Sequencing libraries were prepared on the basis of the Directional mRNA‐Seq Library Prep Pre‐Release Protocol from Illumina (San Diego, USA) according to the manufacturer's instructions.  The RNA-Seq libraries were each sequenced with 76 or 80 cycles in one lane of the Illumina Genome Analyzer IIx platform according to the manufacturer's specifications. After sequencing, we processed the fluorophore intensity files with the standard Illumina base calling algorithms.

tag: tissue/cell type

value: Fibroblast

tag: individual

tag: Sex

value: Male

tag: source

value: Captured in New South Wales, Australia (provided by Adelaide University)

tag: rin

tag: age

value: adult


title: PlatypusOvary

description: Ovulating

data_processing:
For fibroblast libraries: the sequencing files were processed as described in the Brawand, Soumillon, Necsulea et al publication in nature (PMID: 22012392). Briefly, the sequencing reads were mapped to the genome of the platypus (Ensembl annotation 57) using TopHat and Bowtie. A custom script was used to compute RPKM values resolving ambiguities of reads with multiple mapping positions (see original publication for detailed method). The output tab-separated file provides Ensembl IDs of platypus genes (Ensembl annotation 57) and the associated RPKM value (not log-transformed).
For the ovary library: the fastq file was aligned to the platypus genome (Genome and gene annotation: Ensembl 62) using the TopHat software (version 1.0.13) with the following arguments: -p 4 -a 8 -i 40 -I 1000000 -F 0 -m 1 --coverage-search --microexon-search --solexa1.3-quals --butterfly-search --library-type fr-secondstrand. The output (BAM files) was processed by Cufflinks (version 1.0.3) with the following parameters (-p 4 --library-type fr-secondstrand --multi-read-correct --upper-quartile-norm) in order to compute final gene expression levels estimates (FPKM). The provided processed data is presented as a standard cufflinks genes output (http://cufflinks.cbcb.umd.edu/manual.html#tracking_format).
Genome Build:
PlatypusOvaryFemale.txt: Ensembl 62

source_name: Ovary

value: Ovary

value: Female


title: PlatypusFibroblastFemale

description: Ovulating

data_processing:
For fibroblast libraries: the sequencing files were processed as described in the Brawand, Soumillon, Necsulea et al publication in nature (PMID: 22012392). Briefly, the sequencing reads were mapped to the genome of the platypus (Ensembl annotation 57) using TopHat and Bowtie. A custom script was used to compute RPKM values resolving ambiguities of reads with multiple mapping positions (see original publication for detailed method). The output tab-separated file provides Ensembl IDs of platypus genes (Ensembl annotation 57) and the associated RPKM value (not log-transformed).
For the ovary library: the fastq file was aligned to the platypus genome (Genome and gene annotation: Ensembl 62) using the TopHat software (version 1.0.13) with the following arguments: -p 4 -a 8 -i 40 -I 1000000 -F 0 -m 1 --coverage-search --microexon-search --solexa1.3-quals --butterfly-search --library-type fr-secondstrand. The output (BAM files) was processed by Cufflinks (version 1.0.3) with the following parameters (-p 4 --library-type fr-secondstrand --multi-read-correct --upper-quartile-norm) in order to compute final gene expression levels estimates (FPKM). The provided processed data is presented as a standard cufflinks genes output (http://cufflinks.cbcb.umd.edu/manual.html#tracking_format).
Genome Build:
PlatypusFibroblastFemale.txt: Ensembl 57


title: HIV_24hr_rep1

source_name: polyA RNA from HIV-infected SUPT1 at 12 hours post-infection, replicate 2

value: CD4+ human T-cell line SupT1

tag: infection

value: HIV

tag: time


title: 1.5 min (single end)

description: genome build: SGD SAC2


title: HIV_12hr_rep2

source_name: polyA RNA from mock-infected SUPT1 at 12 hours post-infection, replicate 2


title: HIV_12hr_rep1

source_name: polyA RNA from mock-infected SUPT1 at 12 hours post-infection, replicate 1


title: RF_PMM_rep3, MeDIP

city: Ya'an

first: Mingzhou

last: Li

state: Sichuan

address: No.46  Xinkang Road

department: College of Animal Science and Technology

institute: Sichuan Agricultural University

description: MeDIP library for the paired-end sequencing is 50 bp read length.

data_processing: After filtering the low-quality reads, the MeDIP-seq data were aligned to the UCSC pig reference genome (Sscrofa9.2) using SOAP2 (Version 2.21). The differentially methylated regions (DMRs) were identified using our newly developed method by calculating variation of single CpGs. Refer to Methods of the related manuscript for details.

organism: Sus scrofa

source_name: Rongchang pig, psoas major muscle

growth_protocol: Three pig breeds with distinguished fat levels were analyzed:  Landrace (a leaner, Western breed), Rongchang (a fatty, Chinese breed) and Tibetan (a feral, indigenous, Chinese breed that has not undergone artificial selection).

extract_protocol: We randomly selected three female and three male pigs from each breed as biological replicates. We obtained ten tissues for each individual for a total of 180 samples, and sequenced each sample separately. Each MeDIP library was subjected to paired-end sequencing using Illumina HiSeq 2000 and a 50 bp read length. Five ug original DNA was isolated using the E.Z.N.A. HP Tissue DNA Midi Kit (OMEGA) and was sonicated to ~100-500 bp fragments with a Bioruptor sonicator (Diagenode). Then, libraries were constructed using the Illumina Paired-End protocol consisting of end repair, 'A' base addition and adaptor ligation steps, and were performed using Illumina's Paired-End DNA Sample Prep kit following the manufacturer's instructions. Adaptor-ligated DNA was immunoprecipitated using the monoclonal anti-methylcytidine antibody in the Magnetic Methylated DNA Immunoprecipitation Kit (Diagenode). The specificity of the enrichment was confirmed by qPCR using SYBR green mastermix (Applied Biosystems) and primers for positive and negative internal control DNA of non-human samples supplied in the Magnetic Methylated DNA Immunoprecipitation Kit (Diagenode). Cycling of qPCR validation was 95C 5 min, followed by 40 cycles 95C 5 s and 60C 1 min. The enriched fragments with methylation and 10% input DNA were purified on ZYMO DNA Clean & Concentrator-5 columns following the manufacturer's instructions. DNA was eluted in 30 ul buffer EB and its concentration was measured. Enriched fragments were amplified by adaptor-mediated PCR in a final reaction volume of 50 ul consisting of 23 ul purified DNA, 25 ul Phusion DNA polymerase mix (NEB) and 2ul PCR primers. Amplification consisted of 94C 30 s, 10 cycles of 94C 30s, 60C 30 s, 72C 30 s then prolong with 5 min at 72C and products were held at 4C. Amplifications quality and quantity were evaluated by Agilent 2100 Analyzer DNA 1000 chips purified by 2% agarose gel and eluted in 15 ul buffer EB. Ultra-high-throughput 50 bp pair-end sequencing was carried out using the Illumina HiSeq 2000 according to manufacturer's instructions. Raw sequencing data were processed by the Illumina base-calling pipeline.

tag: breed

value: Rongchang

tag: gender

value: female

tag: tissue

value: skeletal muscle:psoas major muscle

tag: medip antibody

value: anti-methylcytidine monoclonal

tag: antibody vendor

value: Diagenode


title: RF_PMM_rep2, MeDIP


title: 20 min (single end) -1

description: genome build: SGD SAC2

value: H4S47C, ∆HHF2, BY4741 (MATa, his3∆1, leu2∆0, met15∆0, ura3∆0)

tag: growth phase

value: Mid-log phase yeast

value: single


title: RF_PMM_rep1, MeDIP


title: RF_LDM_rep3, MeDIP

source_name: Rongchang pig, longissimus doris muscle

value: skeletal muscle:longissimus doris muscle


title: RF_LDM_rep2, MeDIP


title: Mock_12hr_rep3

source_name: polyA RNA from HIV-infected SUPT1 at 24 hours post-infection, replicate 1

tag: cell line

value: Mock

value: 12 hours post-infection


title: HIV_24hr_rep3

source_name: polyA RNA from mock-infected SUPT1 at 24 hours post-infection, replicate 1

value: 24 hours post-infection


title: RF_ULB_rep3, MeDIP

source_name: Rongchang pig, upper layer of backfat

value: adipose:upper layer of backfat


title: RF_ULB_rep2, MeDIP


title: RF_ULB_rep1, MeDIP


title: HIV_12hr_rep3

source_name: polyA RNA from HIV-infected SUPT1 at 12 hours post-infection, replicate 1


title: RF_RAD_rep1, MeDIP

source_name: Rongchang pig, retroperitoneal adipose

value: adipose:retroadipose:peritoneal adipose


title: RF_MAD_rep1, MeDIP

source_name: Rongchang pig, mesenteric adipose

value: adipose:mesenteric adipose


title: RF_PAD_rep2, MeDIP

source_name: Rongchang pig, pericardial adipose

value: adipose:pericardial adipose


title: RF_PAD_rep1, MeDIP


title: RF_ILB_rep1, MeDIP

source_name: Rongchang pig, inner layer of backfat

value: adipose:inner layer of backfat


title: RF_PAD_rep3, MeDIP

value: Rongchang


title: RF_ILB_rep2, MeDIP

value: female


title: RF_IAD_rep3, MeDIP

source_name: Rongchang pig, intermuscular adipose

value: adipose:intermuscular adipose


title: RF_RAD_rep2, MeDIP

value: anti-methylcytidine monoclonal


title: RF_MAD_rep3, MeDIP

value: Diagenode


title: RF_MAD_rep2, MeDIP


title: RF_ILB_rep3, MeDIP


title: RF_IAD_rep1, MeDIP


title: RF_ASA_rep3, MeDIP

source_name: Rongchang pig, abdominal subcutaneous adipose

value: adipose:abdominal subcutaneous adipose


title: RF_ASA_rep2, MeDIP


title: RF_ASA_rep1, MeDIP


title: RM_PMM_rep1, MeDIP

value: male

value: skeletal muscle:psoas major muscle


title: RF_GOM_rep2, MeDIP

source_name: Rongchang pig, greater omentum

value: adipose:greater omentum


title: RF_GOM_rep3, MeDIP


title: RM_LDM_rep1, MeDIP

value: skeletal muscle:longissimus doris muscle


title: RM_PMM_rep2, MeDIP


title: RF_RAD_rep3, MeDIP


title: RM_LDM_rep3, MeDIP


title: RM_LDM_rep2, MeDIP


title: RF_IAD_rep2, MeDIP


title: RF_GOM_rep1, MeDIP


title: RM_ULB_rep3, MeDIP


title: RM_PAD_rep2, MeDIP


title: RM_MAD_rep2, MeDIP


title: RM_ULB_rep2, MeDIP


title: RM_PAD_rep1, MeDIP


title: RM_MAD_rep1, MeDIP


title: RM_ILB_rep3, MeDIP


title: RM_ILB_rep2, MeDIP


title: RM_PAD_rep3, MeDIP


title: RM_ILB_rep1, MeDIP


title: RM_ULB_rep1, MeDIP


title: RF_LDM_rep1, MeDIP


title: RM_RAD_rep2, MeDIP


title: RM_RAD_rep3, MeDIP


title: RM_RAD_rep1, MeDIP


title: RM_MAD_rep3, MeDIP


title: RM_PMM_rep3, MeDIP


title: RM_IAD_rep2, MeDIP


title: RM_GOM_rep2, MeDIP


title: RM_IAD_rep3, MeDIP


title: RM_IAD_rep1, MeDIP


title: RM_GOM_rep3, MeDIP


title: RM_ASA_rep1, MeDIP


title: RM_ASA_rep2, MeDIP


title: RM_GOM_rep1, MeDIP


title: TF_PMM_rep3, MeDIP

source_name: Tibetan pig, psoas major muscle

value: Tibetan


title: TF_LDM_rep2, MeDIP

source_name: Tibetan pig, longissimus doris muscle


title: TF_LDM_rep1, MeDIP


title: TF_PMM_rep2, MeDIP


title: TF_PMM_rep1, MeDIP


title: TF_ULB_rep2, MeDIP

source_name: Tibetan pig, upper layer of backfat


title: TF_LDM_rep3, MeDIP


title: Sclerotinia sclerotiorum sRNA

city: Wuhan

first: Jiasen

last: Cheng

address: Shizishan Street#1

institute: Huazhong Agricultural University

data_processing: Low-quality reads were filtered strictly. 3' adaptor sequences were trimmed and 5' adaptor contaminants were removed. Too small and polyA sequences were removed. Small RNA fragments were compared with the sequences of non-coding RNAs (rRNA, tRNA, snRNA, snoRNA) available in Rfam and the GenBank database. The Sample table includes clean tags with length of 18-30. The supplementary file small.txt includes tags less than 18 nt.

organism: Sclerotinia sclerotiorum

source_name: hypha

growth_protocol: Sclerotinia sclerotiorum were grown at 18-22â on potato dextrose agar (PDA) medium

extract_protocol: The extracted RNA from the four developmental stages of S. sclerotiorum was pooled. The small RNA fraction between 17 and 27 nt was extracted from 15% denaturing polyacrylamide gel. Next, a 5' adaptor and a 3' adaptor were ligated sequentially to 15 ug the small RNAs, and then the short RNAs were converted to DNA by RT-PCR and sequenced on an Illumina-Solexa machine

tag: development stage

value: including four stages:vegetative growth, sclerotial development, myceliogenic germination of sclerotia and pathogenesis of S. sclerotiorum to Arabidopsis thaliana


title: TF_PAD_rep3, MeDIP

source_name: Tibetan pig, pericardial adipose


title: TF_RAD_rep2, MeDIP

source_name: Tibetan pig, retroperitoneal adipose


title: TF_PAD_rep2, MeDIP


title: TF_MAD_rep3, MeDIP

source_name: Tibetan pig, mesenteric adipose


title: TF_ULB_rep1, MeDIP

value: adipose:upper layer of backfat


title: TF_MAD_rep2, MeDIP


title: RM_ASA_rep3, MeDIP


title: TF_MAD_rep1, MeDIP


title: TF_ILB_rep2, MeDIP

source_name: Tibetan pig, inner layer of backfat


title: TF_ILB_rep1, MeDIP


title: TF_GOM_rep3, MeDIP

source_name: Tibetan pig, greater omentum


title: TF_IAD_rep1, MeDIP

source_name: Tibetan pig, intermuscular adipose


title: TF_ULB_rep3, MeDIP


title: TF_IAD_rep2, MeDIP


title: TF_RAD_rep1, MeDIP

value: adipose:retroadipose:peritoneal adipose


title: TF_GOM_rep2, MeDIP


title: TF_GOM_rep1, MeDIP


title: TF_IAD_rep3, MeDIP


title: TF_PAD_rep1, MeDIP


title: TF_ASA_rep3, MeDIP

source_name: Tibetan pig, abdominal subcutaneous adipose


title: TF_RAD_rep3, MeDIP


title: TF_ASA_rep2, MeDIP


title: TF_ILB_rep3, MeDIP


title: TM_PMM_rep1, MeDIP


title: TM_PMM_rep2, MeDIP


title: TM_LDM_rep3, MeDIP


title: TM_ULB_rep1, MeDIP


title: TM_LDM_rep2, MeDIP


title: TM_ULB_rep3, MeDIP


title: TM_PAD_rep2, MeDIP


title: TM_PAD_rep1, MeDIP


title: TM_PAD_rep3, MeDIP


title: TM_LDM_rep1, MeDIP


title: TM_MAD_rep3, MeDIP

value: adipose:mesenteric adipose


title: TM_ILB_rep1, MeDIP


title: TM_ILB_rep2, MeDIP


title: TM_MAD_rep1, MeDIP


title: TM_RAD_rep1, MeDIP


title: TM_IAD_rep2, MeDIP


title: TM_GOM_rep3, MeDIP


title: TM_RAD_rep2, MeDIP


title: TM_IAD_rep1, MeDIP


title: TM_ULB_rep2, MeDIP


title: TM_ILB_rep3, MeDIP


title: TM_GOM_rep1, MeDIP


title: TM_IAD_rep3, MeDIP


title: TM_ASA_rep3, MeDIP


title: TM_PMM_rep3, MeDIP


title: TM_RAD_rep3, MeDIP


title: TF_ASA_rep1, MeDIP


title: TM_MAD_rep2, MeDIP


title: LF_PMM_rep3, MeDIP

source_name: Landrace pig, psoas major muscle

value: Landrace


title: TM_ASA_rep2, MeDIP


title: TM_GOM_rep2, MeDIP


title: LF_PMM_rep1, MeDIP


title: LF_PMM_rep2, MeDIP


title: TM_ASA_rep1, MeDIP


title: LF_ULB_rep3, MeDIP

source_name: Landrace pig, upper layer of backfat


title: LF_LDM_rep2, MeDIP

source_name: Landrace pig, longissimus doris muscle


title: LF_LDM_rep3, MeDIP


title: LF_PAD_rep3, MeDIP

source_name: Landrace pig, pericardial adipose

value: adipose:pericardial adipose


title: LF_RAD_rep1, MeDIP

source_name: Landrace pig, retroperitoneal adipose


title: LF_PAD_rep1, MeDIP


title: LF_LDM_rep1, MeDIP


title: LF_ULB_rep2, MeDIP


title: LF_PAD_rep2, MeDIP


title: LF_RAD_rep2, MeDIP


title: LF_ILB_rep2, MeDIP

source_name: Landrace pig, inner layer of backfat

value: adipose:inner layer of backfat


title: LF_MAD_rep2, MeDIP

source_name: Landrace pig, mesenteric adipose

value: female


title: LF_MAD_rep1, MeDIP


title: LF_MAD_rep3, MeDIP


title: LF_ILB_rep3, MeDIP


title: LF_ILB_rep1, MeDIP


title: LF_IAD_rep1, MeDIP

source_name: Landrace pig, intermuscular adipose

value: adipose:intermuscular adipose


title: LF_IAD_rep2, MeDIP

value: anti-methylcytidine monoclonal


title: LF_GOM_rep3, MeDIP

source_name: Landrace pig, greater omentum

value: Diagenode


title: LF_RAD_rep3, MeDIP


title: LF_ULB_rep1, MeDIP


title: LF_ASA_rep3, MeDIP

source_name: Landrace pig, abdominal subcutaneous adipose

value: adipose:abdominal subcutaneous adipose


title: LF_GOM_rep1, MeDIP


title: LF_ASA_rep1, MeDIP


title: LF_ASA_rep2, MeDIP


title: LM_LDM_rep1, MeDIP

value: male


title: LM_PMM_rep1, MeDIP

value: skeletal muscle:psoas major muscle


title: LM_PMM_rep2, MeDIP


title: LM_LDM_rep3, MeDIP


title: LF_IAD_rep3, MeDIP


title: LM_ULB_rep3, MeDIP


title: LM_ULB_rep2, MeDIP


title: LM_ULB_rep1, MeDIP


title: LM_PAD_rep1, MeDIP


title: LM_PAD_rep2, MeDIP


title: LM_ILB_rep2, MeDIP


title: LM_PAD_rep3, MeDIP


title: LM_RAD_rep1, MeDIP


title: LM_ILB_rep3, MeDIP


title: LM_RAD_rep3, MeDIP


title: LM_MAD_rep2, MeDIP


title: LM_MAD_rep1, MeDIP


title: LM_MAD_rep3, MeDIP


title: LM_RAD_rep2, MeDIP


title: LM_LDM_rep2, MeDIP


title: LM_ILB_rep1, MeDIP


title: LM_IAD_rep3, MeDIP


title: LM_PMM_rep3, MeDIP


title: LM_IAD_rep2, MeDIP


title: LM_IAD_rep1, MeDIP


title: IgG non-dividing rep1

city: Singapore

first: Pierre-Étienne

last: Jacques

address: 60 Biopolis Street

department: Computational and Systems Biology

country: Singapore

institute: Genome Institute of Singapore

data_processing:
Casava v1.7 used for base calling and read mapping.
MACS v1.4.0 used to convert mapped reads into density files (resolution 10bp) using the parameters: --gsize 94093750 --wig -single-profile
In order to be directly comparable, each WIG file was next normalized using the total number of mapped read (per million).
Genome_build: TAIR9
Supplementary_files_format_and_content: WIG files contain the normalized histone density

organism: Arabidopsis thaliana

source_name: Oldest 4 to 6 leaves

growth_protocol: Plants were grown in short day conditions (8h light -16h dark, 20 to 22 °C) for 4 weeks after stratification at 4°C and in dark for 5 days. For harvesting the tissues, we dissected the plants with scalpels under a binocular scope.

extract_protocol: Libraries were constructed according to the guide lines described in the Illumina website. Briefly, 30 µl of chromatin-immuno-precipitated DNA was subjected to the following process: end repair by the addition of a single ‘A’ base, and then ligation of adapters specific for sequencing flow cell. These products were then purified and size-selected on gel to have fragments from 200 to 300bp and enriched by 20 cycles PCR to create the final cDNA library (22 cycles for experiment 1). This library was validated by loading 1 μl of the re-suspended constructs onto an Agilent Technologies 2100 Bioanalyzer DNA-1000 microfluidic chip. The final products showed a distinct band at 200-300bp

tag: strain

value: Columbia HTR13-GFP

tag: chip antibody

value: IgG

tag: genetic background

value: Columbia

value: Oldest 4 to 6 leaves

tag: chip antibody manufacturer

value: Abcam

tag: chip antibody catalog #


title: IgG non-dividing rep2

value: Columbia HTR5-GFP


title: LM_GOM_rep3, MeDIP

value: adipose:greater omentum


title: H3 non-dividing rep2

value: Millipore


title: LM_GOM_rep2, MeDIP

value: Landrace


title: LM_GOM_rep1, MeDIP


title: H3.1 non-dividing rep2

value: GFP

value: Invitrogen


title: LM_ASA_rep2, MeDIP


title: LM_ASA_rep3, MeDIP


title: H3.3 non-dividing rep1


title: LM_ASA_rep1, MeDIP


title: IgG dividing rep1

source_name: Meristem and younger leaves

value: Meristem and younger leaves


title: H3.1 dividing rep2


title: H3 dividing rep2


title: H3 dividing rep1


title: H3.1 dividing rep1


title: LF_GOM_rep2, MeDIP

value: female

value: anti-methylcytidine monoclonal


title: H3.3 dividing rep2


title: H3.3 dividing rep1


title: H3.3 non-dividing rep2


title: RAT04 dividing rep2

data_processing:
Casava v1.7 used for base calling.
TopHat v1.2.0 (using Bowtie v0.12.7 and Samtools v0.1.13) used to map the reads onto TAIR9 (bowtie index file) using recommended settings with optional paramater --max-intron-length 5000
Cufflinks v0.9.3 used with recommended settings to get a FPKM (Fragment Per Kilobase per Million mapped reads) value for each annotation provided (parameter -G with annotation file "TAIR9_GFF3_genes_exons.gtf" dowloaded from ftp://ftp.arabidopsis.org/home/tair/User_Requests/).
Genome_build: TAIR9
Supplementary_files_format_and_content: BAM output files from tophat
Supplementary_files_format_and_content: Tab-delimited text file include FPKM values from each Sample.

extract_protocol: Libraries were constructed according to the guide lines described in the Illumina website. Briefly, 10µg of total RNA was purified to yield poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were copied into first strand cDNA using Superscript II Reverse Transcriptase (Invitrogen) and random primers. This was then followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. These cDNA fragments then underwent an end repair process, the addition of a single ‘A’ base, and then ligation of adapters specific for sequencing flow cell. These products were then purified by gel excision and enriched by PCR with Phusion polymerase (Fermentas) to create the final cDNA library. This library was validated by loading 1 μl of the re-suspended constructs onto an Agilent Technologies 2100 Bioanalyzer DNA-1000 microfluidic chip. The final products showed a distinct band at 200-300bp.


title: RAT03 non-dividing rep2


title: RAT02 dividing rep1


title: RAT01 non-dividing rep1


title: S2_Radicicol_15min

status: Public on May 15 2012

city: Basel

first: Cem

last: Sievers

address: Mattenstrasse

institute: ETH Zurich

data_processing: The number of reads mapping into ensembl genes models were counted using HTSeq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) for subsequent differential expression analysis. Reads were aligned using Bowtie with the following parameter setting: --best --chunkmbs 512 -S -M 100 -p 8 -k 1. Unspecified parameters were set to default values. The dm3 BDGP Release5 was used as reference.

organism: Drosophila melanogaster

source_name: S2 cells

growth_protocol: Schneider medium (Gibco) at 25 degrees celsius

extract_protocol: Exponentially growing Drosophila S2 cells were treated with either DMSO or Hsp90 inhibitor radicicol dissolved in DMSO at 40 micromolar concentration. The treatment was carried out for indicated periods of time  after which cells were harvested, and RNA extracted using TRIzol (invitrogen). Poly(A) RNA were processed for sequencing using Illumina TruSeq manual.

treatment_protocol: Inhibitor added to the medium of growing cells for defined times

tag: cell type

value: cultured cells of embryonic origin

tag: agent

value: Radicicol

tag: time point


title: S2_Radicicol_60min


title: S2_DMSO_Control

value: DMSO


title: S2-DRSC-ChIP-Hsp90_replicate_2

description:
The Hsp90 antibody is not a commercial antibody, but the details were published in the following article:

'Trithorax requires Hsp90 for maintenance of active chromatin at sites of gene expression'. Muhammad Tariq, Ute Nussbaumer, Yujie Chen, Christian Beisel, and Renato Paro. 2009, PNAS.

data_processing:
The Apr. 2006 D. melanogaster genome assembly (dm3, BDGP Release 5) provided by the Berkeley Drosophila Genome Project (BDGP, http://www.fruitfly.org/) was used as a basis for all analyses.  Alignments to the D. melanogaster genome were performed by the software bowtie (Langmead et al. 2009) with parameters '--best --chunkmbs 512 -M 100 -p 8 -k 1 -S'
Supplementary_files_format_and_content: Peak Hsp90 calling results obtained by MACS

source_name: DRSC-S2 cell line

growth_protocol: Drosophila S2-DRSC cells were grown in Schneider's Drosophila Medium (Invitrogen) supplemented with 10 % FBS (Hyclone) at 25°C.

extract_protocol: Exponentially growing S2 cells were resuspended in the growth medium, and cross-linking buffer was added such that the final concentrations in the growth medium were : 5 mM HEPES, 5 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% triton X-100 and 1% formaldehyde. The cells were incubated on end-over rotor at room temperature for 15 min, and the fixation was stopped by adding glycine at a final concentration of 100 mM. The fixed material was spun down and washed twice with cold buffer (50 mM Tris pH 8, 15 mM NaCl, 0.5 mM EGTA, 60 mM KCl). The pellet was resuspended thoroughly in cold sonnication buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA). Sonnication was carried out in cold water in BioRuptor (Diagenode) according to manufacturerâs instructions until a DNA fragment size of 200 â 500 bp was reached. All through the experiments, protease inhibitors were added to cell suspension to avoid any degradation of material.Immunoprecipitation was carried out using anti-Hsp90 antibody (Tariq et al, 2009, PNAS, vol. 106 (4) pp. 1157-62). Libraries for sequencing were prepared with the ChIP-Seq DNA Sample Prep Kit (Cat# IP-102-1001) according to Illumina's instructions. After adapter ligation library fragments of ~250 bp were band isolated from an agarose gel. The DNA was PCR amplified with Illumina primers for 18 (ChIP-seq) cycles, purified and loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.

tag: antibody


title: S2_Radicicol_30min


title: PAR-CLIP CstF-64

first: Andreas

middle: R

last: Gruber

address: Klingelbergstrasse 50-70

department: Biozentrum

institute: University of Basel

description: 3' adapter sequence: TCGTATGCCGTCTTCTGCTTGT

data_processing:
Library strategy: CLIP-Seq
The raw reads were processed on the CLIPZ server (www.clipz.unibas.ch; Khorshid et al., Nucleic Acids Research 2011, 39:D245 (PMID 21087992)).
Reads that appeared to be due to spurious mappings of truncated 3' adaptor sequences (showing a perfect 10-mer match to the 3' adaptor sequence) were discarded. For subsequent analysis, we then extracted uniquely mapped reads with annotations "mRNA", "repeat", or "unknown".
Genome_build: hg19, GRCh37 Genome Reference Consortium Human Reference 37
Supplementary_files_format_and_content: Wiggle files; read densities from PAR-CLIP experiments.

source_name: HEK293, CstF-64 CLIP

growth_protocol: HEK293 cells were routinely grown in DMEM supplemented with 10% FCS in the presence of antibiotics.

extract_protocol: We crosslinked 4-thio-uridine-containing RNA to proteins with 365nm UV light (as in PAR-CLIP) to readily identify crosslinked positions based on the abundant crosslink-diagnostic mutations. We then employed the steps of nuclease digestion, primer ligation and blotting of immunoprecipitated complexes to nitrocellulose from the HITS-CLIP method, to minimize cloning of background RNA. Immunoprecipitation (IP) was performed with protein-specific antibodies or with anti-FLAG antibodies when stably transformed cell lines were generated to produce FLAG-tagged proteins.

tag: crosslinker and crosslinking wavelength

value: Santa Cruz Biotechnology, Inc.

tag: antibody catalog number

tag: antibody lot number


title: PAR-CLIP CPSF-73 rep B

source_name: HEK293, CPSF-73 CLIP


title: PAR-CLIP Fip1

description:
3' adapter sequence: TCGTATGCCGTCTTCTGCTTGT
Antibody: #1005 (lot# 1005-1), PMID 14749727.

source_name: HEK293, Fip1 CLIP

value: not commercial


title: PAR-CLIP CPSF-100 rep B

source_name: HEK293, CPSF-100 CLIP

value: Bethyl


title: PAR-CLIP CstF-64tau rep B

source_name: HEK293, CstF-64tau CLIP


title: PAR-CLIP CPSF-100

description:
3' adapter sequence: TCGTATGCCGTCTTCTGCTTGT
Antibody: MAb-J1/27 (lot# J1/27-1), PMID 7969155.


title: PAR-CLIP CPSF-160 rep B

source_name: HEK293, CPSF-160 CLIP


title: PAR-CLIP Fip1 rep B


title: PAR-CLIP CstF-64tau

tag: genotype/variation

value: stably transformed to produce FLAG-tagged protein

value: monoclonal anti-FLAG M2

value: SIGMA


title: S2-DRSC-ChIP-Hsp90_replicate_1


title: PAR-CLIP CPSF-160

tag: antibody vendor


title: PAR-CLIP CPSF-73


title: PAR-CLIP CPSF-30 repB

source_name: HEK293, CPSF-30 CLIP


title: PAR-CLIP CPSF-30


title: PAR-CLIP CF Im25 rep B

source_name: HEK293, CF Im25 CLIP


title: PAR-CLIP CF Im68 rep B

source_name: HEK293, CF Im68 CLIP


title: PAR-CLIP CF Im59

source_name: HEK293, CF Im59 CLIP


title: A-seq siRNA CstF-64

description: 3' adapter sequence: AAAAAATGGAATTCTCGGGTGCCAAGG

data_processing:
A-seq reads were pre-processed to remove the six diagnostic adenosines derived from the poly(A) tail as well as the 3' adaptor sequence and then were mapped to the human genome (hg19) and annotated using the CLIPZ server. For the construction of the set of 3' end cleavage sites, we only used reads that had the adaptor removed, mapped uniquely to genomic regions and whose annotation was "mRNA", "repeat", "miscRNA", or "unknown". Based on the precise mapping of the 3' ends of these reads we computed putative cleavage sites with their associated abundance. To minimize the frequency of internal priming sites in our data set, we discarded those that in the eight-nucleotide-region immediately downstream of the putative cleavage site had at least seven A nucleotides.
The raw reads were processed on the CLIPZ server (http://http://www.clipz.unibas.ch; Khorshid et al., Nucleic Acids Research 2011, 39:D245).
Genome_build: hg19, GRCh37 Genome Reference Consortium Human Reference 37
Supplementary_files_format_and_content: BED file containing inferred 3' ends

extract_protocol: A-seq protocol: A-seq starts with selection of the poly(A)-containing RNAs on oligo-(dT)25 Dynabeads, followed by partial digestion with RNase I that cleaves after any nucleotide. The 5' ends are then phosphorylated, the 3' ends blocked by 3'dATP and an RNA primer is ligated to the 5' end of the RNA fragments. Our reverse transcription (RT) primer features an anchor nucleotide (A, C or G) designed to align to the first nucleotide upstream of the poly(A) tail, followed by six dTs that anneal to the first six nucleotides (nt) of the poly(A) tail. Following are a stem-loop containing the 3' adaptor sense strand (needed for priming the subsequent PCR reaction), its complement, and finally a stretch of 18 dTs, which together with the 6 dTs after the anchor nucleotide form a 24 nucleotide(nt)-long contiguous oligo dT stretch that aligns to the poly(A) tail. The products of RT and PCR are therefore expected to have 6 As preceding the 3' adaptor (AAAAAATGGAATTCTCGGGTGCCAAGG).

treatment_protocol: For RNAi, HEK293 cells were seeded at a density of 20% in six well plates and all subsequent steps were done according to the "forward method" from the RNAiMAX protocol (Invitrogen). The next day, double stranded siRNAs were incubated with Lipofectamine RNAiMAX (Invitrogen) and added to the wells. After 3 days, cells were harvested and used for confirmation of the siRNA treatment by Western blot and for A-seq. RNA extraction was done with Dynabeads mRNA DIRECT Kit (Invitrogen).

tag: sirna

value: 5'-NNCCUGAAUG GGCGCGAAUUC-3' dsRNA oligo from Dharmacon


title: A-seq siRNA CF Im68

tag: cell line

value: 5'-NNGACCGAGA UUACAUGGAUA-3' dsRNA oligo from Dharmacon


title: A-seq no siRNA


title: A-seq siRNA Ctrl

value: scrambled-A


title: Degradome without AGO1

status: Public on May 14 2012

city: San Francisco

first: David

middle: Eric

last: Weinberg

address: 600 16th Street, GH S572F

institute: University of California, San Francisco

description: Degradome sequencing

data_processing:
After removing the adaptor sequences, reads representing degradome tags were collapsed to a non-redundant set, and 20-21-nt sequences were mapped to the appropriate genome, allowing no mismatches and recovering all hits. The processed files are collapsed to unique sequences (1 line per sequence); the fourth (numeric) value in each column is the number of times each unique sequence was sequenced after removing sequences that did not pass a quality filter; the 2nd column contains the unfiltered read number and the 3rd column contains a "1" if the sequence contains the adaptor sequence 'GTTGGA' and is otherwise blank.
Genome_build: Genome downloaded from the Yeast Gene Order Browser (current as of August 2008)
Supplementary_files_format_and_content: The processed files are text files containing complete 36-nt reads (with adaptor sequences) collapsed to unique sequences with read counts.

organism: Naumovozyma castellii

source_name: Δxrn1 Δago1_RNA-seq

growth_protocol: Yeast strains DPB267 and DPB268 were grown at 25°C in YPD to OD600 0.6.

extract_protocol: Total RNA was isolated using hot phenol from log-phase YPD cultures. Degradome libraries were constructed from 5 μg poly(A)+ RNA as described in Shin et al. (2010).

value: Δxrn1 Δago1

tag: strain id

tag: culture

value: Log-phase liquid culture


title: Degradome with AGO1

description: Degradome sequencing

source_name: Δxrn1 AGO1_RNA-seq

value: Δxrn1 AGO1


title: PAR-CLIP CF Im68


title: PAR-CLIP CF Im25


title: Stam_WI-38_1_4OHTAM_20nM_72hr

data_processing: For data processing details see: http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeUwDnase

growth_protocol: Cells were grown according to ENCODE cell culture protocols: http://genome.ucsc.edu/ENCODE/protocols/cells/human/UW_Stam:WI38_Stam_protocol.pdf

extract_protocol: For extraction protocol details see: http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeUwDnase

tag: cell

tag: cell organism

value: human

tag: cell description

value: Embryonic Lung Fibroblast Cells, hTERT immortalized, includes Raf1 construct

tag: cell karyotype

value: Normal (RAF)

tag: cell lineage

value: Fibroblast

tag: cell sex

value: F

tag: replicate


title: Synovial fibroblast_Tg_2

city: Varkiza

first: Martin

last: Reczko

address: 34 Fleming Street

department: Institute of Molecular Oncology

country: Greece

institute: BSRC A. Fleming

description: <100nt nucleotide fractions isolated from total RNA after being run on a 15% Urea-TBE gel for 1h

data_processing: All reads were aligned using our implementation of a de Bruijn graph representation of all mature miRNAs as defined in mirBase release 15.0.

source_name: Synovial fibroblast

growth_protocol: SFs were isolated from 8-week-old mice by enzymatic digestion of their hind joints. Fibroblasts were selected by continuous culturing for four passages. Cells were grown at 37 8C, 5% CO2 in complete DMEM medium (Gibco-BRL, San Diego, California, United States) supplemented with 10% FBS and 100 U/ml of penicillin-streptomycin.The resulting cultures are >80-90% CD90.2 positive and <1% Mac1 positive, >85% VCAM-1 positive and >80% ICAM-1-positive, as determined by FACS analysis.

extract_protocol: Total RNA was isolated from synovial fibroblasts using the miRVanaâ¢ miR Isolation Kit, Ambion according to the manufactureâs protocol. Libraries were prepared as follows: for the 20-30 and 70-100 nucleotide fractions were isolated from 10ug of total RNA after being run in a 15% Urea-TBE gel (Invitogen) for 1h. The RNA contained on the excised gel bands was eluted on 300Î¼l of 0.3M NaCl solution during 4h at RT and constant rotation. The elute was separated from the gel debris through a Spin-X-column (Fisher) and RNA was precipitated by adding 750Î¼l of 100% ethanol and 3Î¼l of glycogen (Ambion) (1mg/ml) and incubating it for 30min at -80Â°C. The precipitated RNA was centrifuged at 14K rpm for 25 min at 4Â°C, washed with 75% ethanol and resuspended on 5.7Î¼l of RNAse free water. A 5â adaptor was ligated to the RNA in a reaction containing the full amount of RNA recovered from last step, 1.3Î¼l of SRA 5â adaptor (Illumina), 1Î¼l T4 RNA ligase (10U/Î¼l) (Promega), 1 Î¼l 10X T4 RNA ligase (Promega) and 1 Î¼l RNAseOUT (Invitrogen). The reaction was incubated at 20Â°C in a thermal cycler for 6h. The resulting product was run on a 15% Urea-TBE gel (Invitrogen) and the band corresponding to 40-60 nucleotides was excised. The 5â ligated RNA was eluted and precipitated as described before and finally resuspended in 6.4 Î¼l of RNAse free water. A 3â adaptor was added to these molecules in a reaction containing the entire 5â ligated RNA recovered from last step, 0.6 Î¼l of SRA 3â adaptor (Illumina), 1Î¼l T4 RNA ligase (10U/Î¼l) (Promega), 1 Î¼l 10X T4 RNA ligase (Promega) and 1 Î¼l RNAseOUT (Invitrogen). The reaction was incubated at 20Â°C in a thermal cycler for 6h. The product obtained was run on a 10% Urea-TBE gel (Invitrogen) and the band corresponding to 70-90 nucleotides was excised. The 5â and 3â ligated RNA was eluted and precipitated as described before and finally resuspended in 4.5 Î¼l of RNAse free water. In order to synthesize single stranded DNA from this material, 0.5 Î¼l of SRA RT-primer (Illumina) were added to it and the mixture was incubated at 65Â°C for 10 min. This was complemented with 2 Î¼l of 5X first strand buffer, 1 Î¼l 100mM DTT, 0.5 Î¼l RNAseOUT (all from Superscript II reverse transcription kit (Invitrogen) and 0.5 Î¼l 12.5mM dNTP mix (BioLine) and incubated at 48Â°C for 3 min. After adding 1 Î¼l of Superscript II retrotranscriptase (Invitrogen), the reaction was incubated at 44Â°C for 1h. A PCR reaction was then set up with the resultant product and 0.5ul GX1 primer, 0.5ul GX2 primer (both from Illumina), 0.5ul 25mM dNTP mix (Bioline), 10ul 5X cloned Phu buffer, 0.5ul Phu polymerase (both from NEB) and 28ul RNAse free water. The cycling conditions were as follows: 1 cycle of 98Â°C for 30 sec; 15 cycles of 98Â°C for 10 sec, 60Â°C for 30 sec and 72Â°C for 15 sec; 1 cycle of 72Â°C for 10 min. The PCR product was purified by running it on a 10% TBE-PAGE gel for 35 min. The band around 90bp was excised and eluted on 100ul of 1X Elution buffer 2 (NEB) for 2h at RT and at constant rotation. The elute was separated from the gel debris through a Spin-X-column (Fisher) and DNA was precipitated by adding 1Î¼l of glycogen (1mg/ml) (Ambion), 10ul 3M sodium acetate (Ambion) and 325Î¼l of -20Â°C100% ethanol and centrifuging at 14K rpm for 20 min. After a wash with 70% ethanol, the DNA was vacuum dried and resuspended in 10ul of water.

tag: cell type

value: Synovial fibroblast

tag: cell passage

tag: strain

value: CBA/J * C57BL/6/J

value: TghuTNF


title: Synovial fibroblast_Tg_1


title: Synovial fibroblast_WT_1

value: wild type


title: 19-25nt small RNAs associated with recombinant hArgonaute2 purified from SF9 cells

status: Public on May 12 2012

city: Cold Spring Harbor

first: Astrid

last: Haase

state: NY

address: One Bungtown Road

institute: Cold Spring Harbor Laboratory

description:
19-25nt
small RNA cloning by linker ligation

data_processing:
Illumina reads were stripped of the 3’ linker (CTGTAGGCACCATCAATTCGTATGCCGTCTTCTGCTTG) and collapsed to a single representative of each sequence (c1) with multiplicity information retained (absolute read count (c2), reads per million  (RPM) (c3). Reads that matched to synthetic cloning markers as well as low complexity reads were discarded.
Genome_build: n/a
Supplementary_files_format_and_content: processed files are tabular files (txt)  containing sequences, absolute read count, and RPM

organism: Spodoptera frugiperda

source_name: recombinant hArgonaute protein purified from SF9 cells

extract_protocol: RNA was prepared from SF9 cell pellets and immunoaffinity purified hAgo2 using TRIZOL (Invitrogen). 19-29nt long RNAs were PAGE purified from total SF9 RNA. Small RNAs associated with hAgo2 were labeled with 32P gamma-ATP and separated by Urea- PAGE. A size range of 19 to 25 nucleotides was selected, and small RNAs were eluted from the gel. Small RNAs were cloned according to (Brennecke et al., 2007), and sequenced on an Illumina GA2 platform

tag: transgene

value: recombinant hArgonaute

tag: sample type

value: small RNA associated with immunoaffinity purified hAgo2


title: Mec-2_RRBS

status: Public on May 11 2012

city: Augusta

first: Huidong

last: Shi

state: GA

department: Georgia Cancer Center

institute: Augusta University

description: DNA methylation analysis using bisulfite sequencing

data_processing:
Alignment: The raw bisulfite sequencing reads were first converted to “unmethylated reads” via the conversion of all Cs to Ts in silico. The unmethylated, converted sequencing reads were then aligned to the two copies of in silico converted human reference genome (hg18) using SOAP2 (http://soap.genomics.org.cn/). The results of both mappings were combined, and any reads mapped against both copies of converted reference were resolved by identifying their strand based on the TCAG ratio and the mapping positions used accordingly
Methylation calculation: The methylation level [(# of methylated)/(# of methylated + # of unmethylated)] was calculated after measuring the amount of methylation (CG->CG) and unmethylation (CG->TG) in the CpG position. Only CpGs that are covered by at least 10 sequencing reads were included.

source_name: Mec-2 CLL cell line

growth_protocol: Primary B-cells were isolated from CLL using Dynal B-cell Isolation Kit (Invitrogen, Carlsbad, CA, USA); Normal B-cell samples were purchased from ALLCells.

extract_protocol: RRBS was performed according to a previously published protocol (Gu et al. 2010; Meissner et al. 2008) with minor modifications. For each sample, 1  g genomic DNA was first digested overnight using 20 units of MspI (New England Biolabs, NEB). The digested DNA was end-repaired and adenylated in a 20  l reaction consisting of 10U of Klenow fragments (exo-, Enzymatics, MA, USA), and 2  l premixed nucleotide triphosphates (1 mM dGTP, 10 mM dATP, 1 mM methylated dCTP). The reaction was incubated at 30 C for 30 min followed by 37 C for an additional 30 min. The methylated Illumina adapters were ligated with adenylated DNA fragments in a 20  l reaction containing 1  l concentrated T4 ligase (Enzymatics) at room temperature for 15 minutes. The ligation products were gel-selected for fragments with insertion sizes of 40 to 120 bp and 120 to 220 base pairs as previously suggested (Gu et al. 2010; Meissner et al. 2008).  Bisulfite treatment of the sequencing library was conducted using the EZ DNA methylation kit (Zymo Research, Irvine, CA) according to manufacturer's protocol. The final libraries were generated by PCR amplification of 5 ?m of bisulfite-converted template using PfuTurbo Cx Hotstart polymerase and Illumina pair-end PCR primers (PE1.0 and PE2.0).

tag: tissue

value: Blood

value: CD19+ B-cells

tag: tumorgenecity

value: Tumor


title: Mec-1_RRBS

source_name: Mec-1 CLL cell lines


title: MBC_RRBS

source_name: Normal Memory B-cells

value: Normal


title: 19-29-nt small RNAs from SF9 cells

description:
19-29nt
small RNA cloning by linker ligation

source_name: cell line SF9

tag: cell line

value: control

value: small RNA


title: CLL131401_RRBS

source_name: CLL primary B-cells


title: CLL131601_RRBS


title: CLL031001_RRBS


title: CLL030701_RRBS


title: CLL130901_RRBS


title: Synovial fibroblast_WT_2

tag: genotype/variation


title: PMM

description: Tissue from three females were pooled.

data_processing:
The reads that passed through a series of additional filters are called "mappable reads", which are sequences that satisfy the following criteria from the statistics of mammalian miRNAs in miRBbase 16.0: (1) not sequencing adapters; (2) containing no more than 80% A, C, G, or T; (3) containing no more than two N (undermined bases); (4) containing not only A and C or G and T, (5) containing no stretches of A7, C8, G6, or T7; (6) being longer than 14 nt and shorter than 27 nt; (7) not containing more than nine repeats of any dimer, more than five repeats of any trimer, or more than four repeats of any tetramer; (8) been observed more than two times; and (9) not originating from porcine known classes of RNAs (i.e., mRNA in NCBI database; rRNA, tRNA, snRNA and snoRNA in Rfam database; and repetitive sequence elements in Repbase database). All identical reads were counted and combined into one kind.

The UCSC pig reference genome (~2.26 Gbp) (Sscrofa9.2; ftp://hgdownload.cse.ucsc.edu/goldenPath/susScr2/) was used for mapping using NCBI Local BLAST, which included several major steps:
(a) map the mappable reads to known porcine and other mammalian pre-miRNAs from miRBase 16.0 in order;
(b) map the mapped reads in step (a) to the pig genome to obtain their genomic locations and annotations;
(c) cluster the unmapped sequences in step (b) (which are mapped to miRBase sequences, but not to the pig genome) as potential novel miRNAs from the gaps of the genome; and
(d) hairpin RNA structures encompassing reads not-mapped to miRBase in step (a) were predicated from the adjacent 60 nt sequences in either direction using UNAFold complying with criteria from the statistics of mammalian pre-miRNAs in miRBbase 16.0. To avoid the reads being assigned to the ambiguous multiple positions in the pig genome, herein, only the reads longer than 18 nt in length been included.

source_name: psoas major muscle

extract_protocol: Eight different adipose tissues, two phenotypically distinct skeletal muscles, and cardiac muscle of three female Landrace pigs (a leaner, Western breed) were used for small RNA sequencing. Total RNA was extracted using the mirVana miRNA isolation kit (Ambion) following the manufacturer's procedure. For each tissue, equal quantities (5ug) of small RNA isolated from three female Landrace pigs were pooled. Approximately 15 ug of small RNA representing each tissue was used for library preparation and sequencing. The small RNA fraction between 10-40 nt was isolated by polyacrylamide gel electrophoresis (PAGE) and ligated with proprietary adaptors (Illumina). The short RNAs were then converted to cDNA by RT-PCR and each small RNA library was sequenced individually using the Illumina Genome Analyzer II according to manufacturer instructions.

tag: breed

value: Landrace

tag: gender

value: female

tag: developmental stage

value: adult

tag: age

value: 210 days

value: skeletal muscle:psoas major muscle


title: CLL030601_RRBS


title: CLL030501_RRBS


title: CLL131301_RRBS


title: CLL121709_RRBS


title: CLL030801_RRBS


title: CLL030401_RRBS


title: Crystalline KpAGO

description: Small RNA sequencing, 8-24 nt size selection.

data_processing:
After removing the adaptor sequences, applying a quality filter, and removing marker sequences, reads representing the small RNAs were collapsed to a non-redundant set. For analyses of genome-mapping reads, 15-24 nt sequences were mapped to the appropriate plasmid/genome, allowing no mismatches and recovering all hits. The processed files are complete high-quality 36-nt reads (with adaptor sequences) collapsed to unique sequences (1 line per sequence); the second (numeric) value in each column is the number of times each unique sequence was sequenced.
Genome_build: E. coli BL21(DE3) genome (GenBank accession number CP001509)
Supplementary_files_format_and_content: The processed files are text files containing complete 36-nt reads (with adaptor sequences) collapsed to unique sequences with read counts.

organism: Vanderwaltozyma polyspora

source_name: Crystalline K. polysporus Ago1(207-1251) recombinantly expressed in E. coli

growth_protocol: Protein was overexpressed in E. coli BL21(DE3) Rosetta2 (Novagen).

extract_protocol: Total RNA was isolated by phenol extraction, and small-RNA cDNA libraries were prepared as described in Nakanishi et al. (2012).

treatment_protocol: Protein was purified and crystallized as described in Nakanishi et al. (2012).

tag: enzyme expressed

value: K. polysporus Ago1(207-1251)

tag: form

value: Crystalline


title: Soluble KpAGO

source_name: Soluble K. polysporus Ago1(207-1251) recombinantly expressed in E. coli

value: Soluble


title: Wac-3_RRBS

source_name: WacCD5 CLL cell lines


title: NBC_RRBS

source_name: Normal Naïve B-cells


title: early maturation-stage cotyledon adaxial parenchyma compartment

city: Los Angeles

first: Bob

last: Goldberg

address: 610 Charles E Young Drive East

department: Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

description: Cotyledon adaxial parenchyma compartment was captured from ten micron paraffin sections using the Leica LMD6000 system.

data_processing:
Basecalls were performed using RTA version 1.12.4.2.
Original data files from the Illumina sequencing pipeline were in the QSEQ format. Each lane of sequencing was in an individual file.
We aligned the raw reads to a pre-processed reference genome using BS Seeker [Chen et al. BMC Bioinformatics (2010)] allowing for two mismatches. The pre-processed reference genome consisted of sequences from the soybean genome (Glyma version 1.0.1) [Schmutz et al. Nature (2010)] obtained from the Phytozome website (http://phytozome.net), soybean chloroplast genome (GenBank: DQ317523), lambda reference genome (GenBank: J02459), and Phi-X174 reference genome (GenBank: J02482).
Reads containing three consecutive methylation in the non-CG sites were removed, possibly representing non-converted cytosines (Cokus et al. Nature 2008). Clonal reads possibly arising during the PCR amplification step were collapsed into one read.
Methylation level of sampled cytosine was calculated as (methylated calls / (methylated calls + unmethylated calls)).
Genome_build: Glyma version 1.01
Supplementary_files_format_and_content: Tab-delimited text file including methylation level of each sampled cytosine. Column definitions are included in the supplementary README.txt file that is linked to the Series record.

organism: Glycine max

source_name: Laser-captured cotyledon adaxial parenchyma compartment from early-maturation stage cotyledons

growth_protocol: Soybean plants (Williams 82) were grown under standard greenhouse conditions (Le et al., PNAS 2010). Seeds with length between 6.0 and 7.0 mm containing early maturation-stage embryos were harvested.

extract_protocol: Harvested seeds were fixed in 3:1(v/v) ethanol:acetic acid and embedded in paraffin (Kerk et al.,  Plant Physiol.132. 27-35 (2003)).  Seed compartment was captured from ten-micron sections using a Leica LMD6000.  Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA).  Approximately 300 nanogram of genomic DNA was subjected to library preparation following the methods of Hsieh et al. (Hsieh T-F et al. 2009. Science 324:1451-1454) with modifications.  We spiked-in ~ three nanogram of unmethylated lambda DNA (Promega) to serve as a control for complete bisulfite conversion.  Adapter-ligated genomic DNA was subjected to two rounds of bisulfite (BS) treatment using the EpiTect kit (Qiagen, Valencia, CA).  BS-treated DNA was purified using AMpure XP beads (Beckman) and PCR-amplified for 10 cycles using ExTaq (EpiCentre) DNA polymerase.  PCR-amplified DNA fragments were size selected using the AMpure XP beads (Beckman).  Phi-X174 DNA was spiked in to the library by the sequencing facility before cluster formation and sequencing.

tag: cultivar

value: Williams 82

value: early maturation stage

value: cotyledon adaxial parenchyma


title: CD19_RRBS

source_name: Normal CD19+ B-cells

value: Blood


title: Early maturation-stage embryonic cotyledons

description: Embryonic cotyledons from seeds 6.0 - 7.0 mm in length were collected.

data_processing:
Basecalls performed using RTA version 1.12.4.2
Original data file from the Illumina sequencing pipeline.  Each lane of sequencing is attached as an individual compressed file.  The sequence data are in the QSEQ format
We aligned the raw reads to a pre-processed reference genome using BS Seeker [Chen et al. BMC Bioinformatics (2010)] allowing for two mismatches.  The pre-processed reference genome consisted of sequences from the soybean genome (Glyma version 1.0.1) [Schmutz et al. Nature (2010)] obtained from the Phytozome website (http://phytozome.net), soybean chloroplast genome (GenBank: DQ317523), lambda reference genome (GenBank: J02459), and Phi-X174 reference genome (GenBank: J02482).
Reads containing three consecutive methylation in the non-CG sites were removed, possibly representing non-converted cytosines (Cokus et al. Nature 2008). Clonal reads possibly arising during the PCR amplification step were collapsed into one read.
Methylation level of sampled cytosine was calculated as (methylated calls / (methylated calls + unmethylated calls)).
Genome_build: Glyma version 1.01
Supplementary_files_format_and_content: tab-delimited text file including methylation level of each sampled cytosine

source_name: Soybean early maturation-stage embryonic cotyledons

growth_protocol: Soybean plants (Williams 82) were grown under standard greenhouse conditions (Le et al., PNAS 2010).  Whole seeds containing early-maturation (EM) stage embryos were collected. The embryo was dissected out of the seed coat and the axis was manually separated from the cotyledons of the EM-stage embryo.

extract_protocol: Collected tissues were quickly frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Genomic DNA  was isolated from the powder using the DNEASY Plant Mini kit (Qiagen, Valencia, CA) according to manufacturer’s instructions.Approximately one microgram of genomic DNA was subjected to library preparation following the methods of Hsieh et al. (Hsieh T-F et al. 2009. Science 324:1451-1454) with modifications. We spiked-in ~ three nanogram of unmethylated lambda DNA (Promega) to serve as a control for complete bisulfite conversion. Two libraries were constructed from genomic DNA isolated from seed coat. One library was spiked in ~ three nanogram of unmethylated lambda DNA as all other libraries; the other library was spiked in ~ three nanogram of methylated lambda DNA to serve as a control for over conversion (i.e. conversion of methylated cytosine to uracil) during the bisulfite conversion step. The methylated lambda DNA was generated by treating unmethylated lambda DNA with CpG Methyltransferase (New England BioLabs). Adapter-ligated genomic DNA was subjected to two rounds of bisulfite (BS) treatment using the EpiTect kit (Qiagen, Valencia, CA).  BS-treated DNA was purified using AMpure XP beads (Beckman) and PCR-amplified for 10 cycles using ExTaq (EpiCentre) DNA polymerase.  PCR-amplified DNA fragments were size selected using the AMpure XP beads (Beckman).  Phi-X174 DNA was spiked in to the library by the sequencing facility before cluster formation and sequencing.


title: Celniker/RNA:11 extraction1_seq1 aliquote 1

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

institute: Ontario Institute for Cancer Research

data_processing: sequence analysis protocol. 1. 3' adapter sequences starting with 'CTGTAGG' were trimmed with fastx_clipper (fastx_toolkit: http://hannonlab.cshl.edu/fastx_toolkit/). Only reads with recognized 3' adapter and longer than 18 bases were considered in further analysis.2. The trimmed sequences were mapped to genome with Bowtie. only sequences can perfectly match to genome were acceptable.  Processed data are obtained using following parameters: genome version is 5

growth_protocol: Summary of conditions used to produce cell lines for transcriptomics group, excluding W1-Cl.8+, Kc167, S2-DRSC and DmBG3-c2, each of which have their own protocols.  For details see relevant pages at the DGRC Cell Lines page (http://dgrc.cgb.indiana.edu/cells).

extract_protocol:
Protocol for isolation of total RNA from cell cultures, which retains short RNAs.
mRNAs were purified from total RNA extracts, using standard Illumina protocol.  Briefly, 10ug total RNA was bound to oligo(dT) beads, washed twice, and eluted with 10mM Tris-HCl, using Ambion kit.
A Gel purification of RNA sample1. Spike 50 ?g of total RNA with 32P-labeled 19bp and 24bp oligos (~10,000 counts per second) and load the sample in a 15% polyacrylamide/urea gel (Sequagel, National Diagnostics). Run the gel in 1x TBE at constant 10W for 1-2 hours. Expose the gel for 10 minutes in Phosphoimager.2. Excise the band corresponding to the desired size of small RNA (~19-24 nt) into Eppendorf tubes, including the radio-labeled oligos. Add 400?L of 0.4M NaCl. Freeze rapidly in dry ice. Incubate (thaw) for 16 hours at room temperature with agitation to maximize RNA elution.3. Spin down gel slice homogenate through micropore filter (Ultrafree-MC, 0.22?m, Millipore) for 1 minute at room temperature. Add 20?g of glycogen and 2 volumes of 100% Ethanol (RNase-free). Incubate at -20oC for 4 hours.4. Spin the sample at 4oC for 30 minutes. Wash the pellet with 70% EtOH, air dry for 2 min and resuspend in DEPC MQ water and proceed to section B.B 3'-linker ligation and gel purification of the ligated RNA product5. Ligate 50 pmol of Modban oligo (IDT:miRNA cloning linker 1) to the 3? terminus of the purified RNA sample in 1X ATP-free T4 RNA ligase buffer (50mM Tris-HCl (pH 7.5-7.6), 10mM MgCl2, 10mM DTT, 60?g/mL BSA) and 10% DMSO, with 5-10 pmol of T4 RNA ligase 2 Rnl2(1-249) (Ho et al, 2004). Incubate at room temperature for 1 hour 30 min. 6. Load the sample on a 15% polyacrylamide/urea gel as described above and expose the gel for 30 minutes.7. Excise the band corresponding to the desired size of small RNA (~36-41 nt) into Eppendorf tubes, including the ligated radio-labeled oligos.8. Proceed again with overnight elution and precipitation as in steps 2-4.C 5?-linker ligation and gel purification of the ligated RNA product.9. Ligate 50 pmol of Solexa linker to the 5? terminus of the RNA sample in 1XT4 RNA ligase buffer (Ambion) and 10% DMSO with 5-10 Units of T4 RNA ligase (Ambion). Incubate at 37oC for 1 hour 30 min.10. Load the sample on a 15% polyacrylamide/urea gel as described above and expose the gel for 1 hour.11. Excise the band corresponding to the desired size of small RNA (~68-73 nt) into Eppendorf tubes, including the ligated radio-labeled oligos.12. Proceed again with overnight elution and precipitation as in steps 2-4.D Reverse transcription and cDNA amplification.13. Reverse-transcribe the ligated RNA product by using 21 pmol of BanOne primer and 1 ?l Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ?l of reaction. Then, amplify 5 ?l of the cDNA product with 100 pmol of primers Sol_5_SBS3 and Sol_3_Modban, and 5 Units of Taq Polymerase (New England Biolabs): [94oC 15 sec; 54oC 30 sec; 72oC 30 sec] x 5 cycles and [94oC 15 sec; 60oC 30 sec; 72oC 30 sec] x 17 cycles.14. Clean up the PCR product with phenol:chloroform and chloroform. Precipitate the amplified cDNA with Ethanol 100%.E Digestion of the spikes and gel-purification of the amplified cDNA.15. Perform PmeI (New England Biolabs) digestion of radiolabeled oligos (spikes) for 3-4 hours at 37oC and purify the amplified cDNA in a 2% low-melt agarose gel (SeaKem).16. Excise the ~ 120 bp DNA band and proceed to hot-phenol-based gel purification. Transfer aqueous phase to a new eppendorf tube and clean up with phenol:chloroform and chloroform. Precipitate the DNA with Ethanol 100% and resuspend it in 20?L water. 17. Perform Agilent test and dilute the sample to 10nM for Solexa sequencing.Required oligosModban: AMP-5?p=5?pCTGTAGGCACCATCAATdideoxyC-3?  IDT ? Product name: miRNA cloning linker 1      Solexa linker: 5'-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrC                                                   rUrCrUrUrCrCrGrArUrC-3'  IDT ? Custom oligo ? HPLC purified      BanOne primer: 5?-ATTGATGGTGCCTACAG-3?                                                IDT ? Custom oligo ? HPLC purified      Sol_5_SBS3 primer: 5'-AATGATACGGCGACCACCGAACACTCTTTCCCT                                                  ACACGACG-3'  IDT ? Custom oligo ? Desalt      Sol_3_Modban primer: 5'-CAAGCAGAAGACGGCATACGATTGATGGTGCCT ACAG-3'  IDT ? Custom oligo ? HPLC purified
1. cluster generation2. the length of short reads, i.e., the number of sequencing cycles, should list here. in this protocol, it is 35 cycles of sequencing.3. after each cycle, a CCD camera will scan the cells to make a image.

value: embryo-derived cell-line

value: late embryonic stage

tag: Sex

value: Unknown


title: Early maturation-stage embryonic axis

description: Embryonic axis from seeds 6.0 - 7.0 mm in length were collected.

source_name: Soybean early maturation-stage embryonic axis part