description:
design description: MRE-Seq analysis of breast luminal epithelial cell from RM071 (HS2368) using Illumina Genome Analyzer IIx
library name: HS2368
EXPERIMENT_TYPE: DNA Methylation
MRE_PROTOCOL: Refer to document 'Triple-MRE-seq library construction protocol v5' from the Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
MRE_PROTOCOL_CHROMATIN_AMOUNT: 3 ug
MRE_PROTOCOL_RESTRICTION_ENZYME: HpaII, Hin6I, AciI
MRE_PROTOCOL_SIZE_FRACTION: 175-400 bp
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

data_processing:
**********************************************************************

ANALYSIS FILE NAME: GSM613826_UCSF-UBC.Breast_Luminal_Epithelial_Cells.MRE-Seq.RM071.bed
ANALYSIS ALIAS: HS2368-1_705UHAAXX-HS2368-1_70144AAXX-HS2368-1_622VFAAXX-HS2368-1_61GB9AAXX-HS2368-1_624UKAAXX.hg19.level.1
ANALYSIS TITLE: Mapping of Breast Luminal Epithelial Cells MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by MRE-Seq on Breast Luminal Epithelial Cells, Donor RM071 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4207
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 1
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 1 position it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 97.88
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 39

**********************************************************************

ANALYSIS FILE NAME: GSM613826_UCSF-UBC.Breast_Luminal_Epithelial_Cells.MRE-Seq.RM071.wig
ANALYSIS ALIAS: HS2368-1_705UHAAXX-HS2368-1_70144AAXX-HS2368-1_622VFAAXX-HS2368-1_61GB9AAXX-HS2368-1_624UKAAXX.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of Breast Luminal Epithelial Cells MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina MRE-Seq read mappings from Breast Luminal Epithelial Cells, Donor RM071 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4179
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 0bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: BLEC MRE 71
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD Breast Luminal Epithelial Cells MRE-Seq Donor RM071 EA Release 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 97.88
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 39

**********************************************************************


title: salm_inf_2 (s_2)

status: Public on Oct 26 2010

type: SRA

city: Amsterdam

first: Oliver

last: Stockhammer

state: NH

address: Meibergdreef 15

department: Medical Microbiology

country: Netherlands

institute: AMC

description: The total RNA of three biological samples was pooled using equal amounts of RNA. The library was sequenced using 3 pmol of cDNA.

library_source: transcriptomic

data_processing: Image analysis, base calling, extraction of 17 bp tags and tag counting were performed using the Illumina pipeline. Tag counts from duplicate libraries were merged in silico.

molecule: total RNA

organism: Danio rerio

source_name: pooled whole zebrafish embryos injected with Salmonella typhimurium

growth_protocol: Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5-30°C in egg water (60µg/ml Instant Ocean sea salts).

extract_protocol: Tag library preparation was performed using the DGE: Tag Profiling for NlaIII Sample Prep kit from Illumina according to the manufacturer’s instructions. In brief, 1 g of total RNA was used for mRNA capture using magnetic oligo(dT)beads. First- and second-strand cDNA was synthesized and bead-bound cDNA was subsequently digested with NlaIII. Fragments other than the 3 cDNAfragments attached to oligo(dT) beadswerewashed away and a GEX NlaIII adapter was ligated to the free 5 -end of the digested bead-bound cDNA fragments. The GEX NlaIII adapter contains a restriction site for MmeI which cuts 17–18 bp downstream from the NlaIII site, thereby releasing 21–22 bp tags starting with the NlaIII recognition sequence, CATG. A second adapter (GEX adapter 2) was ligated at the site of MmeI cleavage, and the adapter-ligated cDNA tags were enriched using PCR-primers that anneal to the adaptorends. The resulting 85 bp fragments were purified from a 6% acrylamide gel. Purity and yield were checked by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies)

treatment_protocol: Zebrafish embryos were infected with Salmonella typhimurium (strain SL1027) by microinjection of DsRED-labeled bacteria into the caudal vein close to the urogenital opening after the onset of blood circulation (27 hours post fertilization). Samples were collected at 8 hours post injection and immediately quenched in liquid nitrogen and stored at -80 ˚C prior to RNA extraction. For the duration of bacteria injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma). The total RNA of three biological samples was pooled using equal amounts of RNA

tag: tissue

value: whole organism

tag: age

value: 35 hours post fertilization


status: Public on Oct 28 2010

city: San Francisco

first: UCSF-UBC

last: CENTER

state: CA

address: UCSF-UBC

country: USA

institute: UCSF-UBC

description:
design description: MRE-Seq analysis of peripheral mononuclear cell from TC007 (HS2627) using Illumina Genome Analyzer IIx
library name: HS2627
EXPERIMENT_TYPE: DNA Methylation
MRE_PROTOCOL: Refer to document 'Triple-MRE-seq library construction protocol v5' from the Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
MRE_PROTOCOL_CHROMATIN_AMOUNT: 3 ug
MRE_PROTOCOL_RESTRICTION_ENZYME: HpaII, Hin6I, AciI
MRE_PROTOCOL_SIZE_FRACTION: 167-384 bp
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

library_source: genomic

data_processing:
**********************************************************************

ANALYSIS FILE NAME: GSM613898_UCSF-UBC.Peripheral_Blood_Mononuclear_Primary_Cells.MRE-Seq.TC007.bed
ANALYSIS ALIAS: HS2627-1_705WMAAXX-HS2627-1_70601AAXX.hg19.level.1
ANALYSIS TITLE: Mapping of Peripheral Blood Mononuclear Primary Cells MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by MRE-Seq on Peripheral Blood Mononuclear Primary Cells, Donor TC007 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4206
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 1
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 1 position it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 99.15
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 71

**********************************************************************

ANALYSIS FILE NAME: GSM613898_UCSF-UBC.Peripheral_Blood_Mononuclear_Primary_Cells.MRE-Seq.TC007.wig
ANALYSIS ALIAS: HS2627-1_705WMAAXX-HS2627-1_70601AAXX.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of Peripheral Blood Mononuclear Primary Cells MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina MRE-Seq read mappings from Peripheral Blood Mononuclear Primary Cells, Donor TC007 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4178
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 0bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: PBM MRE 07
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD Peripheral Blood Mononuclear Primary Cells MRE-Seq Donor TC007 EA Release 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 99.15
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 71

**********************************************************************

molecule: genomic DNA

organism: Homo sapiens

extract_protocol: library construction protocol: Refer to document 'Triple-MRE-seq library construction protocol v5' from the Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)

tag: submitted sample id

tag: Sex

value: female

tag: body site

value: Blood

tag: histological type

value: Peripheral blood mononuclear cell

tag: is tumor

value: No

value: primary cell


title: salm_inf_1 (s_1)

description: The total RNA of three biological samples was pooled using equal amounts of RNA. The library was sequenced using 2 pmol of cDNA.


title: Twi12low native IP

city: Boston

first: Mary

middle: T

last: Couvillion

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

description:
small RNA sequencing ("Hannon" primer)
library strategy: CLONEEND
library selection: RT-PCR, PCR
Library strategy and selection is described in the reference above.

data_processing:
The 36nt long Illumina reads were stripped of the 3’ linker and the resulting small RNA sequences were mapped to the annotated tRNA and rRNA regions of the sequenced Tetrahmyena macronuclear genome, assembly 2. Only reads matching the  genome 100%, or with a 3' mismatch of _C, _CC, or _CCA, because of the posttranscriptional modification of mature eukaryotic tRNA, were used for further analysis. seqlen = the small RNA sequence length, in nucleotides. mapnum = the number of times the small RNA matches the respective genome sequence. _C, _CC, or _CCA indicates untemplated nucleotides added to the sequence shown.  All small RNAs were converted into DNA for sequencing, therefore U is represented as T.
Note - "mappers" here include only sequences matching tRNA or rRNA.

organism: Tetrahymena thermophila

source_name: cells

growth_protocol: media: 1XSPP, 30 degrees C.

extract_protocol:
Small RNA libraries were prepared as described in:
Couvillion MT, Lee SR, Hogstad B, Malone CD, Tonkin LA, Sachidanandam R, Hannon GJ, Collins K. 2009. Sequence, biogenesis, and function of diverse small RNA classes bound to the Piwi family proteins of Tetrahymena thermophila. Genes Dev 23: 2016-2032.
Gel-purified sRNAs  were first ligated to 50 pmol pre-adenylated 3' adaptor (IDT-miRNA cloning linker 1) using truncated T4 RNA Ligase 2 (NEB) in 50 mM Tris-HCl at pH 7.5, 10 mM MgCl2, 10 mM DTT, and 60 'g/mL BSA. Ligated products were resolved, SYBR Gold stained, and gel purified as described above, then ligated to 50 pmol RNA 5' adaptor using T4 RNA ligase (Ambion) in buffer provided and 1/10 volume DMSO. Ligated products were again gel-purified and reverse transcribed with SuperScript II (Invitrogen) and RT primer. First strand cDNA was amplified with Taq polymerase using RT primer and PCR primer, with an annealing temperature of 54°C for 5 cycles and 60°C for 14 cycles. After phenol:chloroform extraction and ethanol precipitation, products were separated from primers on a 2% low-melt agarose gel. Products were retrieved by melting agarose slices in 0.3 M NaCl and extracting with warmed phenol, phenol:chloroform, then chloroform, followed by ethanol precipitation.
3' adaptor (IDT-miRNA cloning linker 1):
5'-rAppCTGTAGGCACCATCAATdideoxyC-3'
5' adaptor:
5'-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrC-3'
RT primer:
5'-CAAGCAGAAGACGGCATACGATTGATGGTGCCTACAG-3'
PCR primer:
5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACG-3'
IgG IP (Rabbit IgG-Agarose is from Sigma, catalog number A2909) from S100 or S16 extract. Native IPs were done using S100, crosslinked IPs using S16.

treatment_protocol: No cadmium (transgene expression is uninduced, allowing only leaky expression).

tag: strain

value: mZZTwi12 (CU522)

tag: library

value: sRNAs associated with slightly overexpressed ZZ-tagged Twi12 purified under native conditions (size range 15-34nt)


title: Twi12low xlink IP

value: sRNAs associated after formaldehyde crosslinking (size range 15-22nt)


description:
design description: MeDIP-Seq analysis of breast myoepithelial cell from RM066 (HS2375) using Illumina Genome Analyzer IIx
library name: HS2375
EXPERIMENT_TYPE: DNA Methylation
EXTRACTION_PROTOCOL: Refer to document 'MeDIP-seq library construction protocol v2' by Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR: Diagenode Bioruptor
EXTRACTION_PROTOCOL_SONICATION_CYCLES: 25 min
MeDIP_PROTOCOL: Refer to document 'MeDIP-seq library construction protocol v2' by Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
MeDIP_PROTOCOL_DNA_AMOUNT: 2 ug
MeDIP_PROTOCOL_BEAD_TYPE: Protein A/G Beads
MeDIP_PROTOCOL_BEAD_AMOUNT: 100 ul
MeDIP_PROTOCOL_ANTIBODY_AMOUNT: 1 ug
MeDIP_ANTIBODY: Methylcytidine
MeDIP_ANTIBODY_PROVIDER: Eurogentec
MeDIP_ANTIBODY_CATALOG: BI-MECY-0100
MeDIP_ANTIBODY_LOT: 80604
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

data_processing:
**********************************************************************

ANALYSIS FILE NAME: GSM613857_UCSF-UBC.Breast_Myoepithelial_Cells.MeDIP-Seq.RM066.bed
ANALYSIS ALIAS: HS2375-1_612Y6AAXX-HS2375-1_61WRCAAXX.hg19.level.1
ANALYSIS TITLE: Mapping of Breast Myoepithelial Cells MeDIP-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by MeDIP-Seq on Breast Myoepithelial Cells, Donor RM066 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4265
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: MeDIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 1
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 1 position it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_MEDIP-SEQ_EXPERIMENTS_SCORED: 24
FINDPEAKS_SCORE: 0.0273
FINDPEAKS_PERCENTILE: 4
HOTSPOT_SCORE: 0.1854
HOTSPOT_PERCENTILE: 8
IROC_SCORE: 0.8031
IROC_PERCENTILE: 12
POISSON_SCORE: 0.6591
POISSON_PERCENTILE: 25

**********************************************************************

ANALYSIS FILE NAME: GSM613857_UCSF-UBC.Breast_Myoepithelial_Cells.MeDIP-Seq.RM066.wig
ANALYSIS ALIAS: HS2375-1_612Y6AAXX-HS2375-1_61WRCAAXX.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of Breast Myoepithelial Cells MeDIP-Seq Data
ANALYSIS DESCRIPTION: Illumina MeDIP-Seq read mappings from Breast Myoepithelial Cells, Donor RM066 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4241
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MeDIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 200bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: BMC MeDIP 66
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD Breast Myoepithelial Cells MeDIP-Seq Donor RM066 EA Release 2

QUALITY SCORES:
NUMBER_OF_MEDIP-SEQ_EXPERIMENTS_SCORED: 24
FINDPEAKS_SCORE: 0.0273
FINDPEAKS_PERCENTILE: 4
HOTSPOT_SCORE: 0.1854
HOTSPOT_PERCENTILE: 8
IROC_SCORE: 0.8031
IROC_PERCENTILE: 12
POISSON_SCORE: 0.6591
POISSON_PERCENTILE: 25

**********************************************************************

extract_protocol: library construction protocol: Refer to document 'MeDIP-seq library construction protocol v2' from the Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)

value: Breast

value: Myoepithelial cell

value: breast myoepithelial cell


description:
design description: MRE-Seq analysis of peripheral cd4 naive from TC003 (HS2378) using Illumina Genome Analyzer IIx
library name: HS2378
EXPERIMENT_TYPE: DNA Methylation
MRE_PROTOCOL: Refer to document 'Triple-MRE-seq library construction protocol v5' from the Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
MRE_PROTOCOL_CHROMATIN_AMOUNT: 1.5 ug
MRE_PROTOCOL_RESTRICTION_ENZYME: HpaII, Hin6I, AciI
MRE_PROTOCOL_SIZE_FRACTION: 170-400 bp
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

data_processing:
**********************************************************************

ANALYSIS FILE NAME: GSM613842_UCSF-UBC.CD4_Memory_Primary_Cells.MRE-Seq.TC003.bed
ANALYSIS ALIAS: HS2378-1_622VFAAXX-HS2378-1_61GB9AAXX.hg19.level.1
ANALYSIS TITLE: Mapping of CD4 Memory Primary Cells MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by MRE-Seq on CD4 Memory Primary Cells, Donor TC003 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4201
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 1
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 1 position it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 95.8
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 17

**********************************************************************

ANALYSIS FILE NAME: GSM613842_UCSF-UBC.CD4_Memory_Primary_Cells.MRE-Seq.TC003.wig
ANALYSIS ALIAS: HS2378-1_622VFAAXX-HS2378-1_61GB9AAXX.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of CD4 Memory Primary Cells MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina MRE-Seq read mappings from CD4 Memory Primary Cells, Donor TC003 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4173
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 0bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: CD4M MRE 03
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD CD4 Memory Primary Cells MRE-Seq Donor TC003 EA Release 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 95.8
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 17

**********************************************************************

value: CD4 naive cell


title: Piwi-IP_squ_het_ovaries

status: Public on Oct 25 2010

city: Cold Spring Harbor

first: Astrid

last: Haase

state: NY

address: One Bungtown Road

institute: Cold Spring Harbor Laboratory

description: 19-29nt small RNA

data_processing: The 3' linker (CTGTAGGCACCATCAATTCGTATGCCGTCTTCTGCTTG) was clipped from the sequences, which are then collapsed and mapped perfectly to the Drosophila genome (dm3, release 5). Annotation tracks (RefSeq genes, miRBase miRNAs and RepeatMasker) were used to assign annotations to mapped sequences. The analysis of small RNA libraries was essentially performed as described in Czech et al. 2009 (Czech, B., Zhou, R., Erlich, Y., Brennecke, J., Binari, R., Villalta, C., Gordon, A.,Perrimon, N., and Hannon, G.J. 2009. Hierarchical rules for Argonaute loading in Drosophila. Mol Cell 36(3): 445-456).

organism: Drosophila melanogaster

source_name: Piwi immunoprecipitates from squ_het [squHE47/CyO, squPP32/CyO] ovaries

extract_protocol: RNA was isolated from total ovaries and Piwi immunoprecipitates with Trizol, then separated PAGE to obtain a small RNA fraction ranging from 19 to 29 nt. Adapters for Solexa sequencing were ligated, then the ligated product amplified by RT-PCR. The PCR product is gel-purified for sequencing

value: ovaries

tag: developmental stage

value: 4-5 day old adult flies

tag: genotype/variation

value: [squHE47/CyO, squPP32/CyO]


title: Piwi-IP_squ_mut_ovaries

description: 19-29nt small RNA

source_name: Piwi immunoprecipitates from squ_mut [squHE47/PP32] ovaries


title: Piwi-IP_zuc_het(H-Y)_ovaries

description: 19-29nt small RNA

source_name: Piwi immunoprecipitates from zuc_het [zucHM27/CyO, zucSG63/CyO] ovaries

value: [zucHM27/CyO, zucSG63/CyO]


title: Piwi-IP_zuc_het(mut)_ovaries

description: 19-29nt small RNA

source_name: Piwi immunoprecipitates from zuc_het [zucHM27/CyO, zucDf(2I)PRL/CyO] ovaries

value: [zucHM27/CyO, zucDf(2I)PRL/CyO]


title: salm_contr_2 (s_5)

source_name: pooled whole zebrafish embryos injected with PBS

treatment_protocol: Zebrafish embryos were infected with PBS by microinjection into the caudal vein close to the urogenital opening after the onset of blood circulation (27 hours post fertilization). Samples were collected at 8 hours post injection and immediately quenched in liquid nitrogen and stored at -80 ˚C prior to RNA extraction. For the duration of bacteria injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma). The total RNA of three biological samples was pooled using equal amounts of RNA

value: whole organism

value: 35 hours post fertilization


title: zuc_H-Y_ovaries

description: 19-29nt small RNA

source_name: 19-29-nt small RNA from zuc catalytically dead (H-->Y) [zucHM27/SG63] ovaries


title: 50-75 nt piR-ILs associated with Armitage from OSCs

city: Shinjuku-ku

first: Kuniaki

last: Saito

state: Tokyo

address: 35 Shinanomachi

country: Japan

institute: Keio University School of Medicine

description:
small RNAs associated with Armitage
Armi-associated, small RNA (50-75 nt) sequencing

data_processing: The raw data files were parsed by our custom perl script. Small RNA sequences were mapped to the Drosophila melanogaster draft assembly (Berkeley Drosophila Genome Project release 5) using the NCBI BLASTN program. The annotation of each small RNA was determined by examining the overlap between its mapped regions and other feature track data of the UCSC Genome Browser (http://genome.ucsc.edu/).

source_name: Ovarian somatic cell (OSC) line

growth_protocol: OSCs  were grown at 25°C

extract_protocol: Armi was immunopurified from OSCs using a specific antibody. After immunoprecipitation, total RNAs were isolated from the anti-Armi immunoprecipitates. Acidic dephosphorylation was carried out with T4 polynucleotide kinase (Takara) in 50 mM Tris-HCl (pH 6.5), 10 mM MgCl2 and 5mM dithiothreitol. Enzymes were inactivated by phenol/chloroform extraction, followed by RNA precipitation. Precipitates were dissolved in H2O. RNAs were resolved on a 12 % denaturing polyacrylamide gel with 5’-labeled Decade Marker (Ambion) as a size marker. 50-75 nt RNAs were cut out from the gel. Cloning of RNAs was carried out using standard protocols. The sequencing was performed on a GS FLX system (Roche).

value: adult

tag: cell type

value: Ovarian somatic cell

tag: antibody

value: anti-Armi antibody

tag: antibody details

value: mouse monoclonal antibody raised by our laboratory


title: N2_L2_ssRNALigSeq

status: Public on Oct 20 2010

city: Stanford

first: Ayelet

last: Lamm

address: 300 Pasteur Drive, Lane Bldg Room L302

department: Pathology

institute: Stanford University School of Medicine

data_processing: The processed data files in fasta format contain the parsed RNA reads for each sample (without 5' barcode sequence). The raw files each correspond to a single lane of sequence sequences. The 5’ barcode was trimmed from the barcoded samples and the homopolymer A tail was trimmed from all reads that were obtained using the CircLigSeq method. Identical sequences were collapsed in each sample to avoid PCR "jackpots".

molecule: polyA RNA

organism: Caenorhabditis elegans

source_name: whole L2 hermaphrodite

extract_protocol: single-strand-capture, double-strand-capture, and circligase-based RNA-seq

value: whole L2 hermaphrodite

tag: genotype

value: wild type

tag: rna-seq method

value: ssRNALigSeq

tag: gender

value: hermaphrodite


title: N2_L4_ssRNALigSeq

source_name: whole L4 hermaphrodite

tag: tissue

value: whole L4 hermaphrodite


title: Piwi-IP_zuc_mut_ovaries

description: 19-29nt small RNA

source_name: Piwi immunoprecipitates from zuc_mut [zucHM27/Df(2I)PRL] ovaries


title: N2_L3_CircLigSeq

source_name: whole L3 hermaphrodite

value: whole L3 hermaphrodite

value: CircLigSeq


title: N2_L2_CircLigSeq


title: N2_mixed-stage_polysomes

source_name: Mixed-stage hermaphrodite

value: whole mixed-stage hermaphrodite

value: mixed stage


title: S2_MAGO-2

city: Montreal

first: Hugo

last: Lavoie

state: Quebec

address: 6100 Royalmount

country: Canada

institute: CNRC-IRB

description: Transcriptome of S2 cells treated with MAGO dsRNA; replicate 2.

data_processing: Reads were mapped using ABI Bioscope 1.0.1 to both the Drosophila melanogaster genome (UCSC dm6) and a set of filter sequences, comprised of ribosomal and transfer RNA and sequencing adapters.  Filtered reads are not removed from the results but simply marked as such.  Both .bam and .wig files were generated using the Whole Transcriptome pipeline described in the ABI documentation.  A read was considered unique even if other matches existed of length 3 less than the best match (clear zone = 3).  The .wig file was generated using the default values for the sam2wig run (alignment score of 24 or higher and minimum coverage of 10).

source_name: S2 cell culture, MAGO dsRNA

growth_protocol: S2 cells were grown in Schneider Medium (Invitrogen) with 10% FBS and with Pen/Strep at 18C degrees.

extract_protocol: Total RNA from S2 cells subjected to treatment with eIF4AIII, mago and GFP dsRNA in duplicate was prepared using TRIzol reagent (Invitrogen). Poly(A)+ mRNAs were enriched using oligo(dT) selection with the Oligotex mRNA Midi kit (Qiagen). The resulting mRNA was then depleted of rRNA molecules using the Ribominus Eukaryote kit (Invitrogen). High-throughput sequencing libraries were prepared according to the SOLiD whole transcriptome library preparation protocol (Applied Biosystems).

treatment_protocol: S2 cells were treated with dsRNAs (15ug/ml) corresponding to either GFP, eF4AIII or MAGO for six days.

tag: cell line

tag: treatment

value: MAGO dsRNA

tag: treatment time

value: 6 days


title: salm_contr_1 (s_3)

value: whole organism

tag: age


title: S2_MAGO-1

description: Transcriptome of S2 cells treated with MAGO dsRNA; replicate 1.


title: zuc_het(H-Y)_ovaries

description: 19-29nt small RNA

source_name: 19-29-nt from zuc heterozygous (het) [zucHM27/CyO, zucSG63/CyO] ovaries

value: ovaries

value: 4-5 day old adult flies

value: [zucHM27/CyO, zucSG63/CyO]


title: N2_L1_CircLigSeq

source_name: whole L1 hermaphrodite

value: whole L1 hermaphrodite


title: rrf-3_him-8_ssRNALigSeq

source_name: whole adult male

value: whole adult male

value: adult

value: male


status: Public on Oct 21 2010

description:
design description: MRE-Seq analysis of H1 embryonic stem cell line (HS1473) using Illumina Genome Analyzer IIx
library name: HS1473
EXPERIMENT_TYPE: DNA Methylation
MRE_PROTOCOL: Refer to document 'Triple-MRE-seq library construction protocol v5' from the Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
MRE_PROTOCOL_CHROMATIN_AMOUNT: N/A ug
MRE_PROTOCOL_RESTRICTION_ENZYME: MspI
MRE_PROTOCOL_SIZE_FRACTION: 186-428 bp
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

data_processing:
**********************************************************************

ANALYSIS FILE NAME: GSM543005_UCSF-UBC.H1.MRE-Seq.Batch2.2.wig
ANALYSIS ALIAS: UCSF-UBC.H1.MRE-Seq.Batch2.2
ANALYSIS TITLE: Raw Signal Density Graphs of H1 Cell Line MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina MRE-Seq read mappings for DNA methylation assessment in the H1 Cell Line were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.2859
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MRE-Seq
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 0bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None

**********************************************************************

ANALYSIS FILE NAME: GSM543005_UCSF-UBC.H1.MRE-Seq.HS1473.bed
ANALYSIS ALIAS: HS1473-1_6005PAAXX-HS1473-1_42TE4AAXX.hg19.level.1
ANALYSIS TITLE: Mapping of H1 Cell Line MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by MRE-Seq on the H1 Cell Line, Library HS1473 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4217
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 1
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 1 position it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 90.19
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 10

**********************************************************************

ANALYSIS FILE NAME: GSM543005_UCSF-UBC.H1.MRE-Seq.HS1473.wig
ANALYSIS ALIAS: HS1473-1_6005PAAXX-HS1473-1_42TE4AAXX.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of H1 Cell Line MRE-Seq Data
ANALYSIS DESCRIPTION: Illumina MRE-Seq read mappings from the H1 Cell Line, Library HS1473 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.4189
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MRE-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 0bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: H1 MRE 73
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD H1 Cell Line MRE-Seq Library HS1473 EA Release 2

QUALITY SCORES:
NUMBER_OF_MRE-SEQ_EXPERIMENTS_SCORED: 28
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS: 90.19
PERCENT_READS_MAPPING_TO_RESTRICTION_FRAGMENTS_PERCENTILE: 10

**********************************************************************

tag: submitted sample id

value: cell line

tag: line

tag: Sex

tag: body site

value: Embryo ICM

tag: histological type

value: Embryonic stem cell

value: No


title: Piwi-IP_zuc_H-Y_ovaries

description: 19-29nt small RNA

source_name: Piwi immunoprecipitates from zuc_H-Y [zucHM27/SG63] ovaries

tag: developmental stage

tag: genotype/variation


title: S2_eIF4AIII-2

description: Transcriptome of S2 cells treated with eIF4AIII dsRNA; replicate 2.

source_name: S2 cell culture, eIF4AIII dsRNA

value: eIF4AIII dsRNA


title: him-8_ssRNALigSeq

tag: strain

value: ssRNALigSeq


title: S2_eIF4AIII-1

description: Transcriptome of S2 cells treated with eIF4AIII dsRNA; replicate 1.


title: him-8_dsDNALigSeq

value: dsDNALigSeq


title: fem-1_dsDNALigSeq

source_name: whole adult hermaphrodite

value: whole adult hermaphrodite

value: hermaphrodite


title: S2_GFP-2

description: Transcriptome of S2 cells treated with a control GFP dsRNA; replicate 2.

source_name: S2 cell culture, GFP dsRNA

value: GFP dsRNA

value: 6 days


title: S2_GFP-1

description: Transcriptome of S2 cells treated with a control GFP dsRNA; replicate 1.


title: fem-3_dsDNALigSeq


title: N2_mixed-stage_dsDNALigSeq

value: whole mixed-stage hermaphrodite

value: wild type

value: mixed stage


title: Control_Input_DNA_sequencing_for_PRDM14

city: Singapore

first: Yuriy

middle: L

last: Orlov

address: 60 Biopolis Street

department: Computational Systems Biology

country: Singapore

institute: Genome Institute of Singapore

description: control input DNA sequencing, no IP

data_processing: Alignment: Sequence reads were obtained and mapped to the human (hg18) genome using the Illumina Genome Analyzer II Pipeline. All uniquely mapped reads with two or fewer mismatches were retained.

source_name: Embyonic stem cells

growth_protocol: The hESC line H1 (WA-01, passage 28) was used for this study. They were cultured feeder free on matri-gel (BD). Condition medium used for culturing human contained 20% KO serum replacement, 1 mM L-glutamine, 1% non-essential amino acids and 0.1 mM 2-mercaptoethanol and an additional 8ng/ml of basic fibroblast growth factor (Invitrogen) supplemented to the hESCs unconditioned medium. Medium was changed daily.

extract_protocol: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with PRDM14 antibody.  Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: No treatment was performed for ESCs used for ChIP-sequencing.

tag: cell type

value: pluripotent and self-renewing cells

tag: antibody

value: none, control input DNA


title: N2_L3_ssRNALigSeq

value: whole L3 hermaphrodite

tag: genotype

tag: rna-seq method


title: Human ES cells replicate 1 - size Fraction 5

city: St Lucia

first: Sean

last: Grimmond

state: Q

address: Services Rd

department: Institute for Molecular Bioscience

country: Australia

institute: University of Queensland

description: mRNA from human ES cells selected for size (6500bp and greater)

data_processing: Raw data processed with Bioscope 1.2

source_name: human ES cells

growth_protocol: HES2 cells were cultured as per Kolle et al., Stem Cells 2009

extract_protocol: SOLiD Whole transcriptome analysis Kit - RNA fragmented to ~100bp, RNA-DNA hybrid adaptors ligated, cDNA made, PCR amplified and gel purified

treatment_protocol: Cells were treated with cycloheximide for 10 minutes prior to harvesting (Kolle et al., 2009)

tag: selected size

value: 6500bp and greater


title: Human ES cells replicate 1 - size Fraction 4

description: mRNA from human ES cells selected for size (3500-6500bp)


title: N2_L1_ssRNALigSeq

tag: tissue

value: whole L1 hermaphrodite

tag: gender


title: PRDM14_ChIPSeq

description: Chromatin IP against PRDM14

data_processing:
Alignment: Sequence reads were obtained and mapped to the human (hg18) genome using the Illumina Genome Analyzer II Pipeline. All uniquely mapped reads with two or fewer mismatches were retained.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/) using processed data file. Control input DNA sequencing library was used as background for peak calling.

value: custom made PRDM14


title: Human ES cells replicate 1 - size Fraction 2

description: mRNA from human ES cells selected for size (500-2000bp)


title: Human ES cells replicate 1 - size Fraction 3

description: mRNA from human ES cells selected for size (2000-3500bp)


title: Human ES cells replicate 1 - size Fraction 1

description: mRNA from human ES cells selected for size (0-500bp)


title: mRNA-Seq

status: Public on Oct 19 2010

city: Chicago

first: Dali

last: Han

state: IL

address: 5801 South Ellis Avenue

department: Department of Chemisty

institute: University of Chicago

description:
mRNA sequencing raw reads from post-mortem superior frontal gyrus from five male individuals of 8,9,10,11 and 14
For the mRNA-seq, the average insert size is 150nt and the standard deviation is 50 nt.

data_processing:
For the H3K4me3 Chip-Seq and Input aligned file, it is in the bed format. Procressed by Soap2 with default parameters. Then filter the duplicate reads.
For the mRNA Sequenceing aligned file, it is in the bedgraph format. Procressed by Tophat with following parameters: "-r 100 -a 8 -m 0 -g 100 --solexa1.3-quals --coverage-search --microexon-search --segment-mismatches 2 --segment-length 25"
For the peak calling file,The meaning of each clolumn is listed below:
Column 1: Chromosome number
Column 2: Start
Column 3: End
Column 4: FDR
The peak calling use SICER with following parameters: w=200 g=200 FDR cutoff=1e-3.

organism: Macaca mulatta

source_name: prefrontal cortex

extract_protocol:
The rhesus macaque samples were obtained from the Suzhou Experimental Animal Center (Suzhou, China). Chromatin from the 9-year old frozen rhesus macaque prefrontal cortex tissue was fragmented by sonication and immunueprecipitated by the incubation with antibody against H3K4me3 (cat. ab8580), then DNA was purified for the next library construction. Fragmented genomic DNA without the immunueprecipitation procedure was purified for the next similar treatment as control. Sequencing tags of 36-mer were obtained from the Solexa single-end pipeline. Total RNA was extracted from the dissected frozen prefrontal cortex tissue using Trizol® reagent (Invitrogen, Carlsbad, CA) from five male individuals of 8, 9, 10, 11 and 14 years old and equal amount of them were pooled together. Low molecular weight RNA was isolated, ligated to the adapters, amplified, and sequenced following the paired-end sample preparation protocol (Illumina, USA) and sequenced as 75-mers X2 using the Illumina 1G Genome Analyzer paired-end pipeline.
Library construction was following Illumina's protocol.

value: Male


title: H3K4me3_ChipSeq

description: H3K4me3 Chip-sequencing raw reads from post-mortem superior frontal gyrus of a 3389 day old rhesus individual

data_processing:
For the H3K4me3 Chip-Seq and Input aligned file, it is in the bed format. Procressed by Soap2 with default parameters. Then filter the duplicate reads.
For the mRNA Sequenceing aligned file, it is in the wig format. Procressed by Tophat with following parameters: "-r 100 -a 8 -m 0 -g 100 --solexa1.3-quals --coverage-search --microexon-search --segment-mismatches 2 --segment-length 25"
For the peak calling file,The meaning of each clolumn is listed below:
Column 1: Chromosome number
Column 2: Start
Column 3: End
Column 4: FDR
The peak calling use SICER with following parameters: w=200 g=200 FDR cutoff=1e-3.

value: 3389 days

tag: antibody catalog

tag: antibody vendor

value: Abcam


title: ML-DmD9 V029/BS102 (Celniker/RNA:73) rep2 extraction2_seq1 aliquote 1

status: Public on Oct 18 2010

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 mapped and trimmed, allowing 2 mismatches when starting from base 20 in a read. Only reads with recognized 3' adapter and longer than 18 bases were considered in further analysis.2. The trimmed sequences were mapped to genome by BLAT. only sequences can perfectly match to genome were acceptable.  Processed data are obtained using following parameters: genome version is 5

source_name: ML-DmD9 cell small ncRNA RNA-Seq

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 sample.  1. Spike 50 ug 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 400uL 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, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug 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 product.  5. 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, 60ug/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 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul 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 20uL water.  17. Perform Agilent test and dilute the sample to 10nM for Solexa sequencing.
1. cluster generation  2. 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.

tag: cell line

value: dorsal mesothoracic disc

value: third instar larval stage

value: y v f mal

value: Unknown


title: Input

description: Input DNA sequencing raw reads from post-mortem superior frontal gyrus of a 3389 day old rhesus individual

tag: age


title: Lai_CantonS_ovary extraction1_seq1 aliquote 1

growth_protocol:
Adult Growth Collection protocol;  Young adult flies were fed with fly food supplemented with yeast for 3-4 days. Agef flies were anesthetized with CO2 and collected in an eppendorf tube. The flies were frozen on dry ice and stored at –80C until use. [edit]
Ovary dissection protocol;  Feed young adult flies with yeast supplemented food for 3-4 days. Dissect the flies in PBS using two pairs of forceps. Keep the dissected ovaries in ice-cold PBS. Discard PBS and freeze the samples on dry ice. [edit]

extract_protocol:
1. For every 100ul tissue, homogenize in an eppendorf tube using   plastic pestle (Kimble-Chase 749521-1590) in 100ul of TRIzol LS   (Ambion). After homogenization add 800ul TRIzol LS and 100ul of   distilled water. Add 200ul chloroform and mix the sample by vortex.2. Spin the sample for 10 minutes at 14000 rpm and 4oC. 3. Transfer the aqueous phase to a different eppendorf tube and add   500 ul of isopropanol (Sigma). Incubate overnight at -20oC. 4. Spin down the RNA for 15 minutes at 14000 rpm and 4oC. 5. Wash the pellet with 70% Ethanol. 6. Resuspend the RNA pellet in ~30 ul of sterile MQ water.
A Gel purification of RNA sample1. Spike 50 ug 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 400uL 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, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug 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, 60ug/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 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul 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 20uL 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: Canton S

value: Adult ovaries

value: Adult Female

tag: Sex

value: Female


title: ML-DmD32 V035/BS26 (Celniker/RNA:165) rep2 extraction2_seq1 aliquote 1

source_name: ML-DmD32 cell small ncRNA RNA-Seq


title: Lai ML-DmD21 (Celniker/RNA:49, Replicate 1) extraction1_seq1 aliquote 1

source_name: ML-DmD21 (Celniker/RNA:49) extraction1_seq1 channel_1

growth_protocol: Cell Line Growth Others protocol;  Cells from the line LINE (referenced in REF) were obtained from the Drosophila Genomics Resource Center (https://dgrc.cgb.indiana.edu/), and were grown in petri dishes at 25ºC. Each 100 mm tissue culture-grade petri dish contained 10 ml of cells growing in MEDIUM. Cells were maintained between approximately CONC. and 1.0 x 10^7 cells/ml, and harvested at about 5-7.5 x 10^6 cell/ml. Do not allow any of the cells to become overgrown; some cell lines will not recover if overgrown. For additional information about the line, see relevant DGRC page (https://dgrc.cgb.indiana.edu/cells). LINE: 1182-4H REF: Debec, A. (1978). "Haploid cell cultures of Drosophila melanogaster." Nature 274(5668): 255-256 MEDIUM: M3+BPYE + 10% FCS CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: CME-L1 REF: Currie, D. A., Milner, M.J., Evans, C.W. (1988). "The growth and differentiation in vitro of leg and wing imaginal disc cells from Drosophila melanogaster." Development 102: 805-814. MEDIUM: M3+ 2% FCS + 5 µg/ml insulin + 2.5% fly extract CONC.: 2.5 x 10^6 --------------------------------------------------------------------- LINE: CME-W2 REF: Currie, D. A., Milner, M.J., Evans, C.W. (1988). "The growth and differentiation in vitro of leg and wing imaginal disc cells from Drosophila melanogaster." Development 102: 805-814. MEDIUM: M3+ 2% FCS + 5 µg/ml insulin + 2.5% fly extract CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: GM2 REF: Mosna, G., Dolfini, S. (1972). "Morphological and chromosomal characterization of three new continuous cell lines of Drosophila melanogaster." Chromosoma 38: 1-9 MEDIUM: M3+BPYE + 5% FCS CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: mbn2 REF: Gateff, E., Gissmann, L., Shrestha, R., Plus, N., Pfister, H., Schroder, J., Hausen, H.Z. (1980). Characterization of two tumorous blood cell lines of Drosophila melanogaster and the viruses they contain. Invertebrate Systems in Vitro. Fifth International Conference on Invertebrate Tissue Culture, Rigi-Kaltbad, Switzerland, 1979. E. Kurstak, Maramorosch, K., Dubendorfer, A. Amsterdam, Elsevier: 517-533. MEDIUM: M3+BPYE + 10% FCS CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmBG1-c1 REF: Ui, K., Nishihara, S., Sakuma, M., Togashi, S., Ueda, R., Miyata, Y., Miyake, T. (1994). Newly established cell lines from Drosophila larval CNS express neural specific characteristics. In Vitro Cell. Dev. Biol. A. 30(4): 209-216. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmBG2-c2 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui-Tei, K., Nishihara, S., Sakuma, M., Matsuda, K., Miyake, T., Miyata, Y. (1994). Chemical analysis of neurotransmitter candidates in clonal cell lines from Drosophila central nervous system. I. ACh and L-DOPA. Neurosci. Lett 174(1): 85-88. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD11 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD17-c3(Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD16-c3 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD20-c2 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD20-c5 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD21 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD32 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-Dm4-c1 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-Dm4-c1 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD8 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: ML-DmD9 (Stick hard to substrate) Use cell scraper or Pasteur pipette to remove cells REF: Ui, K., Ueda, R., Miyake, T. (1987). "Cell lines from imaginal discs of Drosophila melanogaster." In Vitro Cell. Dev. Biol. A. 23: 707-711. MEDIUM: M3+BPYE + 10% FCS + 10 µg/ml insulin CONC.: 3 x 10^6 --------------------------------------------------------------------- LINE: S1 REF: Schneider, I. (1972). "Cell lines derived from late embryonic stages of Drosophila melanogaster. ." J. Embryol. exp. Morphol. 27: 353-365. MEDIUM: M3+BPYE + 12.5% FCS CONC.: 2.5 x 10^6 --------------------------------------------------------------------- LINE: S2R+ REF: Schneider, I. (1972). "Cell lines derived from late embryonic stages of Drosophila melanogaster. ." J. Embryol. exp. Morphol. 27: 353-365. MEDIUM: M3+BPYE + 10% FCS CONC.: 2 x 10^6 --------------------------------------------------------------------- LINE: S3 REF: Schneider, I. (1972). "Cell lines derived from late embryonic stages of Drosophila melanogaster. ." J. Embryol. exp. Morphol. 27: 353-365. MEDIUM: M3+BPYE + 12.5% FCS CONC.: 2.5 x 10^6 --------------------------------------------------------------------- LINE: Sg4 REF: Schneider, I. (1972). "Cell lines derived from late embryonic stages of Drosophila melanogaster. ." J. Embryol. exp. Morphol. 27: 353-365. MEDIUM: M3+BPYE + 10% FCS CONC.: 2.5 x 10^6 [edit]

extract_protocol:
RNA extraction protocol;  Note: This protocol is used to prepare RNA from cell cultures that will be used for the isolation of microRNAs. Materials * 75% ethanol, made with DNase/RNase-free water. * TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml). * DNase/RNase-free water: Invitrogen cat. no. 10977. * 75% ethanol, prepared with DNase/RNase-free water. Procedure 1. Pellet cells from 5-10 plates by centrifugation in 50 ml disposable polypropylene tubes (approximately 1000 x g, 5 min.). 2. Discard the supernatant. Resuspend the pellet in 5 ml Drosophila PBS; transfer to a 15 ml polypropylene tube. 3. Pellet cells again by centrifugation. 4. Remove supernatant. 5. Lyse the pellet in TRIzol reagent (0.75 ml per 5-10 x 107 cells) by repetitive pipetting. For 5 plates of cells at 5x106 cells/ml, use 2 ml TRIzol. 6. Incubate at room temperature for 5 minutes. 7. Add 0.267 ml chloroform per ml of TRIzol used in step 5. 8. Shake tubes vigorously for 15 seconds. 9. Incubate at room temperature for 2 minutes. 10. Centrifuge for 15 minutes at 4degC at 12,000 x g. 11. Transfer the top (aqueous) phases to clean tubes. 12. Precipitate the RNA from the aqueous phase by adding 0.67 ml of isopropanol per ml of TRIzol used in step 5. 13. Invert tubes once to mix gently. 14. Incubate samples at -20º overnight. 15. Centrifuge for 10 minutes at 4° at 12,000 x g. 16. Remove supernatant. 17. Wash RNA pellet once with 75% ethanol, 0.7 ml per microfuge tube. 18. Vortex briefly. 19. Centrifuge at 7,500 x g for 5 minutes at 4°. 20. Let pellet air dry for 10 minutes. Note: DO NOT let the pellet dry completely. 21. Dissolve in RNase-free water.  22. Incubate at 37° overnight to dissolve the RNA. Determine concentration by absorbance, using a Nanodrop® ND-1000 Spectrophotometer. Store at -80°. [edit]
MRNA purfication from total RNA for Illumina seqencing protocol;  [edit] Standard Illumina mRNA Purification from Total RNA 1. Dilute 10 ug Total RNA nuclease-free H2O to 50µL in a 1.5 mL RNase free non-sticky eppendorf tube (Ambion). 2. Heat at 65°C for 5 minutes to disrupt the secondary structures, and place on ice. 3. Meanwhile, aliquot 100µL of dynal oligo(dT) beads into a 1.5mL RNase free non-sticky eppendorf tube (Ambion). 4. Wash the beads twice with 100µL Binding Buffer. 5. Resuspend the beads in 50µL Binding Buffer, and add the 50µL of total RNA sample from step 1.2; rotate at RT for 5 minutes. 6. Remove the supernatant and wash the beads twice with 100µL of Washing Buffer B. 7. Aliquot 80µL of Binding Buffer to a fresh 1.5mL RNase free non-sticky eppendorf tube (Ambion). 8. Add 20µL of 10mM Tris-HCl to the beads, heat at 800C for 2 minutes to elute mRNA from the beads. Immediately put on the magnet stand and transfer the supernatant (mRNA) to the tube from step 1.7. And add 100µL of Washing Buffer B to the beads. 9. Heat the samples at 65°C for 5 minutes to disrupt the secondary structures, and place on ice. 10. Meanwhile Wash the bead twice with 100µL of Washing Buffer B. 11. Add 100µL of mRNA sample from step 9; rotate at RT for 5 minutes. 12. Remove the supernatant and wash the beads twice with 100µL of Washing Buffer B. 13. Add 10 or 12 µL of 10mM Tris-HCl to the beads, heat at 800C for 2 minutes to elute mRNA from the beads. Immediately put on the magnet stand and transfer the supernatant (mRNA) to a fresh 200µL thin wall PCR tube, and there should be ~9 or 11 µL of mRNA. [edit]
A Gel purification of RNA sample1. Spike 50 ug 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 400uL 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, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug 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, 60ug/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 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul 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 20uL 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.


title: Lai_Kc_Rubin_Ecdysone extraction1_seq1 aliquote 1

growth_protocol:
S2 and S2R+ cell culture:S2 (or S2R+) cells were grown in Schneider's Drosophila medium supplemented with 10% FBS, 100U/ml penicillin and 100ug/ml streptomycin at 26C in 25cm^2 or 75cm^2 flasks. Cells were harvested by centrifugation at 216g for 5 minutes in a 15ml conical tube. The pellet was resuspended in Trizol LS reagent.
Ecdysone exposure protocol;  cells were cultured in standard media supplemented with 5x10[-6] 20-hydroxyecdysone for 48 hours prior to RNA harves [edit]

extract_protocol:
1. Spin down 1 ml of confluent samples for 5 minutes at 2000 rpm. 2. Discard supernatant and resuspend the cells in 100 ul of medium. 3. Add 900 ul of Trizol LS (Ambion) and incubate for 10 minutes at   room temperature. 4. Add 200 ul of chloroform (Sigma) and shake by vortex. 5. Spin the sample for 10 minutes at 14000 rpm and 4oC. 6. Transfer the aqueous phase to a different eppendorf tube and add   500 ul of isopropanol (Sigma). Incubate overnight at -20oC. 7. Spin down the RNA for 15 minutes at 14000 rpm and 4oC. 8. Wash the pellet with 70% Ethanol. 9. Resuspend the RNA pellet in ~30 ul of sterile MQ water.
A Gel purification of RNA sample1. Spike 50 ug 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 400uL 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, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug 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, 60ug/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 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul 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 20uL 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: Kc-Rubin

value: embryo-derived cell-line

value: late embryonic stage

value: se/e


title: Lai_Female_Head_HeatShocked_V007 extraction1_seq1 aliquote 1

growth_protocol:
Adult Growth Collection protocol;  Young adult flies were fed with fly food supplemented with yeast for 3-4 days. Agef flies were anesthetized with CO2 and collected in an eppendorf tube. The flies were frozen on dry ice and stored at –80C until use. [edit]
Heat Shock treatment protocol;  Flies shorted out on day 5th on Day 9th after hatching Three hours in incubator at 36 Celsius degrees Samples frozen in Nitrogen after treatment Storaged at -80 degrees in eppendorf (100 flies per eppendorf) [edit]
1. Freeze 3-5 days old adult flies at -80C 2. Vortex frozen flies in a 15ml conical tube. 3. Sieve heads and bodies by mesh # 20 and  # 40. Heads are recovered   from #40 fraction. Proceed directly to total RNA isolation or freeze   the samples at -80C.

extract_protocol:
Heads and bodies1. Freeze 3-5 days old adult flies at -80C2. Vortex frozen flies in a 15ml conical tube.3. Sieve heads and bodies by mesh # 20 and  # 40. Heads are recovered from #40 fraction and bodies are recovered from #20 fraction. 4. Legs and wings are mostly lost in this procedure.5. Homogenize heads or bodies in an eppendorf tube using plastic pestle (Kimble-Chase 749521-1590) in 100ul TRIzol LS. After homogenization, add 800ul of TRIzol LS and 100ul water. Embryos1. Allow flies to lay eggs on apple juice plates supplemented with yeast paste for the indicated time.2. Wash eggs with distilled water. Collect eggs using nylon mesh.3. Dechorionate eggs by soaking 50% Clorox for 1 minute.4. Wash dechorionated eggs with distilled water.5. Collect eggs in an eppendorf tube. Freeze the samples at -80C. Extraction of total RNAs1. Spin down 1 ml of confluent samples for 5 minutes at 2000 rpm.2. Discard supernatant and resuspend the cells in 100 ul of medium.3. Add 900 ul of Trizol (Ambion) and incubate for 10 minutes at room temperature.4. Add 200 ul of chloroform (Sigma) and shake by vortex.5. Spin the sample for 10 minutes at 14000 rpm and 4oC.6. Transfer the aqueous phase to a different eppendorf tube and add 500 ul of isopropanol (Sigma). Incubate overnight at -20oC.7. Spin down the RNA for 15 minutes at 14000 rpm and 4oC.8. Wash the pellet with 70% Ethanol.9. Resuspend the RNA pellet in ~30 ul of sterile MQ water.
A Gel purification of RNA sample1. Spike 50 ug 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 400uL 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, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug 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, 60ug/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 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul 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 20uL 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: Female heads

value: wild type


title: Lai_Female_Head_Aged_V013 extraction1_seq1 aliquote 1

growth_protocol:
Adult Growth Collection protocol;  Young adult flies were fed with fly food supplemented with yeast for 3-4 days. Agef flies were anesthetized with CO2 and collected in an eppendorf tube. The flies were frozen on dry ice and stored at –80C until use. [edit]
Aging procedure protocol;  Collect living flies when 70% of the population died (in our case, 42 days after hatching) [edit]
1. Freeze 3-5 days old adult flies at -80C 2. Vortex frozen flies in a 15ml conical tube. 3. Sieve heads and bodies by mesh # 20 and  # 40. Heads are recovered   from #40 fraction. Proceed directly to total RNA isolation or freeze   the samples at -80C.


title: Lai_S2_Rubin_Ecdysone extraction2_seq1 aliquote 1

value: Male


title: Lai_Kc_Rubin_Ecdysone extraction2_seq1 aliquote 1


title: Lai_S2_Rubin_Ecdysone extraction1_seq1 aliquote 1


title: V002 extraction1_seq1 aliquote 1

source_name: V002 extraction1_seq1 channel_1

growth_protocol: 1. Allow flies to lay eggs on apple juice plates supplemented with yeast paste for the indicated time. 2. Wash eggs with distilled water. Collect eggs using nylon mesh. 3. Dechorionate eggs by soaking 50% Clorox for 1 minute. 4. Wash dechorionated eggs with distilled water. 5. Collect eggs in an eppendorf tube. Proceed directly to total RNA isolation or freeze the samples at -80C.

extract_protocol:
1. Allow flies to lay eggs on apple juice plates supplemented with yeast paste for the indicated time.2. Wash eggs with distilled water. Collect eggs using nylon mesh.3. Dechorionate eggs by soaking 50% Clorox for 1 minute.4. Wash dechorionated eggs with distilled water.5. Collect eggs in an eppendorf tube. Freeze the samples at -80C. 6. Spin down 1 ml of confluent samples for 5 minutes at 2000 rpm. 7. Discard supernatant and resuspend the cells in 100 ul of medium. 8. Add 900 ul of Trizol LS (Ambion) and incubate for 10 minutes at   room temperature. 9. Add 200 ul of chloroform (Sigma) and shake by vortex. 10. Spin the sample for 10 minutes at 14000 rpm and 4oC. 11. Transfer the aqueous phase to a different eppendorf tube and add   500 ul of isopropanol (Sigma). Incubate overnight at -20oC. 12. Spin down the RNA for 15 minutes at 14000 rpm and 4oC. 13. Wash the pellet with 70% Ethanol. 14. Resuspend the RNA pellet in ~30 ul of sterile MQ water.
A Gel purification of RNA sample1. Spike 50 ug 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 400uL 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, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug 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, 60ug/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 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul 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 20uL 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: 2-18hr embryo


title: Lai_CantonS_Embryo_Irradiated extraction1_seq1 aliquote 1

growth_protocol:
1. Allow flies to lay eggs on apple juice plates supplemented with yeast paste for the indicated time. 2. Wash eggs with distilled water. Collect eggs using nylon mesh. 3. Dechorionate eggs by soaking 50% Clorox for 1 minute. 4. Wash dechorionated eggs with distilled water. 5. Collect eggs in an eppendorf tube. Proceed directly to total RNA isolation or freeze the samples at -80C.
Embryos  were collected overnight (~0-16 hrs) then aged 2 hours to obtain ~2-18 hrs embryos. The embryos were X-irradiated with 4000 rads;After one hour, the embryos were dechorionated, rinsed, and frozen in liquid N2 prior to total RNA extraction.


title: N2_mixed-stage_ssRNALigSeq

value: whole mixed-stage hermaphrodite

value: ssRNALigSeq

value: mixed stage

value: hermaphrodite


title: Lai_Ago2-414_ovary extraction1_seq1 aliquote 1

growth_protocol:
Adult Growth Collection protocol;  Young adult flies were fed with fly food supplemented with yeast for 3-4 days. Agef flies were anesthetized with CO2 and collected in an eppendorf tube. The flies were frozen on dry ice and stored at –80C until use.
Ovary dissection protocol;  Feed young adult flies with yeast supplemented food for 3-4 days. Dissect the flies in PBS using two pairs of forceps. Keep the dissected ovaries in ice-cold PBS. Discard PBS and freeze the samples on dry ice.

extract_protocol:
1. For every 100ul tissue, homogenize in an eppendorf tube using   plastic pestle (Kimble-Chase 749521-1590) in 100ul of TRIzol LS   (Ambion). After homogenization add 800ul TRIzol LS and 100ul of   distilled water. Add 200ul chloroform and mix the sample by vortex.2. Spin the sample for 10 minutes at 14000 rpm and 4oC. 3. Transfer the aqueous phase to a different eppendorf tube and add   500 ul of isopropanol (Sigma). Incubate overnight at -20oC. 4. Spin down the RNA for 15 minutes at 14000 rpm and 4oC. 5. Wash the pellet with 70% Ethanol. 6. Resuspend the RNA pellet in ~30 ul of sterile MQ water.
A Gel purification of RNA sample1. Spike 50 ug 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 400uL 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, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug 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, 60ug/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 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul 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 20uL 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 is 35 cycles of sequencing.3. after each cycle, a CCD camera will scan the cells to make a image.