title: CME L1 (Celniker/RNA:85) extraction1_seq1 aliquote 1

source_name: CME L1 (Celniker/RNA:85) extraction1_seq1 channel_1

growth_protocol: Cell Line Growth Others protocol;  Cells from the 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). : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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

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°.
MRNA purfication from total RNA for Illumina seqencing protocol;   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.
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.

value: ventral prothoracic disc

value: third instar larval stage

value: Unknown


title: Adult Male Body (Replicate 3) extraction3_seq1 aliquote 1

status: Public on Oct 18 2010

type: SRA

city: Toronto

first: DCC

last: modENCODE

state: Ontario

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

country: Canada

institute: Ontario Institute for Cancer Research

library_source: transcriptomic

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

molecule: total RNA

organism: Drosophila melanogaster

growth_protocol: 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. Bodies are recovered   from #20 fraction. Legs and wings are mostly lost in this procedure.   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 is 35 cycles of sequencing.3. after each cycle, a CCD camera will scan the cells to make a image.

tag: strain

value: Canton S

tag: tissue

value: Male body

tag: developmental stage

value: Adult male

tag: genotype

value: wild type

tag: Sex

value: Male


title: Lai_r2d2_ovary_totalRNA 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.

value: Adult ovaries

value: Adult Female

value: Female


title: Tissue:Adult_ovaries:EL:1 extraction1_seq1 aliquote 1_Ago1IP_2744

description: channel ch1 is RIP RNA; Antibody information listed below: official name: Ago1;target name: Ago1;host: Rabbit;antigen: Synthetic peptide derived from within residues 1 - 100 of Fruit fly (Drosophila melanogaster) Ago1.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab5070;reference: http://www.biocompare.com/ProductCompare.aspx?i=304729 http://www.biocompare.com/ProductCompare.aspx?i=470260

growth_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.

extract_protocol:
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. Bodies are recovered   from #20 fraction. Legs and wings are mostly lost in this procedure.   Proceed directly to total RNA isolation or freeze the samples at -80C.
Samples are homogenized in 100ul Hepes-NP40 buffer using plastic pestles and then 900ul Hepes-NP40 is added .Lysates are cleared by centrifugation at 4C for 10 minutes at 10,000g. Cleared extract was split and incubated with rabbit polyclonal AGO1 antibody (1:20; lot number 113754, Abcam) for 4 h at 4 uC. AGO1 antibodies were isolated by adding protein G beads (1:10; Roche) for 1 h. Beads were washed six times each for 10 min in buffer B (30 mM HEPES, pH 7.4, 800 mM NaCl, 2 mM MgCl2, 0.1% NP-40), which contained equal supplements as buffer A. The immunoprecipitation was analysed by western blotting using anti-AGO1 (1:2,000; Abcam). AGO1-associated RNAs were isolated with phenol/chloroform and ethanol precipitated.
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.


title: Lai_Dcr-2_L811fsX_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.


title: embryos,14-24 hr after egg laying (Celniker/RNA:253,259,265,277) extraction1-4

description:
embryos,14-16 hr after egg laying (Celniker/RNA:253) extraction1_seq1 aliquote 1
embryos,16-18 hr after egg laying (Celniker/RNA:259) extraction2_seq1 aliquote 1
embryos,18-20 hr after egg laying (Celniker/RNA:265) extraction3_seq1 aliquote 1
embryos,22-24 hr after egg laying (Celniker/RNA:277) extraction4_seq1 aliquote 1

growth_protocol:
Fly population cages contained the sequenced D. melanogaster isogenic strain with the mutations; yellow (y1), cinnabar (cn1), brown (bw1), and speck (sp1) [1]. Two homemade Plexiglas cages approximately 25cm x 25cm x 25cm, were used for staged embryo collections. These population cages were constructed from ¼ inch solid Plexiglas except for the open front panel. The fronts of the cages were covered with sleeves constructed of muslin cloth that were attached to the Plexiglas sides using Velcro strips. Each muslin sleeve was then twisted and held closed with a hose clamp. Flies were fed with three hard egg lay collection plates made in 150 X 15 mm Petri dishes containing a substrate of 3.3% agar, 13% unsulfured molasses, and 0.15% Tegasept. The hard egg lay plates were completely covered with a thin layer of moist yeast paste (Fleischmann's Baker's Dry Yeast) and placed horizontally on a short 1cm raised Plexiglas bar in the bottom of each cage to avoid crushing flies. The two population cages were maintained at 24 C in a controlled environment on a 24-hour light cycle (14 hours light / 10 hours dark) using three 60W bulbs on a timer. Flies were introduced to the cages at least four days prior to collections and fertility counts. These animals were initially grown in bottles at the same controlled environment described above. Each population cage was established from four trays with 35 bottles per tray containing newly eclosed adult flies. Each 250ml (pint) bottle contained approximately 40ml standard Drosophila medium in use at Bloomington (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/media-recipes.htm). The isogenic Drosophila mutant strain was propagated in bottles (A) starting with approximately 40-50 adults per bottle on day zero. New bottles (B) were generated from these bottles (A) on day 14. Cages were established by clearing the 'A' bottles into an empty bottle (without food) and transferring the newly eclosed adult flies in the 'A' bottles into the two population cages on day 18. The next round of cages and bottles (C) were established from the 'B' bottles in the same manner. Two-hour embryo collections were made during the light cycles following at least one two-hour pre-lay.
This protocol was used for staging and collection of Drosophila melanogaster embryos by Dave Miller(Kaufman Lab, IU Bloomington) for transcriptome analysis by the Celniker group.

extract_protocol:
Protocol for isolation of insect RNA using Trizol.  This protocol is used to prepare RNA that will be used for the isolation of microRNAs.  [TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml)]
mRNAs were purified from total RNA extracts, using standard Illumina protocol.  Briefly, 10ug total RNA was bound to oligo(dT) beads, washed twice, and eluted with 10mM Tris-HCl, using Ambion kit.
A Gel purification of RNA sample1. Spike 50 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.

value: Y cn bw sp


title: ML-DmBG3-c2 V076/BS179 (Celniker/RNA:121) rep1 extraction1_seq1 aliquote 1

source_name: ML-DmBG3-c2 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: CNS-derived cell-line

value: y v f mal


title: CME W1 Cl.8+ (Celniker/RNA:59) extraction2_seq1 aliquote 1

source_name: CME W1 Cl.8+ (Celniker/RNA:59) extraction2_seq1 channel_1

growth_protocol: Cell Line Growth CME W1 protocol;  CME W1-Cl.8+ Source: DGRC, stock #151. M3 medium + 2% heat-inactivated FCS + 5 μg/ml insulin + 2.5% fly extract. * The source of insulin is probably not critical, but should be kept constant in case it matters. Purchase a sterile 10 mg/ml solution of human insulin from Sigma (cat. no. I9278); this can be stored for at least 6 months at 4° C. Dilute 1:2000 in medium + FCS + fly extract. The complete medium is good for about a month at 4° C, with the fly extract being the most unstable component. * Fly extract is prepared according to the protocol given on the DGRC web-site (https://dgrc.cgb.indiana.edu/files/tissue-culture-medium-additions.pdf). You may make the extract yourself, or you request it from the Cherbas lab. As a courtesy, we will supply fly extract at no cost to members of the modENCODE consortium, but you will be asked to supply a Fedex number to pay for the shipment. You will need 2.5 ml per 100 ml of medium. Tubes of fly extract can be stored for at least 1 year at -20° C. Thawing the tube requires no special precautions. To order fly extract, send e-mail to Lucy Cherbas (dgrc@cgb.indiana.edu) . Thawing cells As soon as possible after you receive them, thaw an ampoule of frozen cells in a 25 cm2 T-flask, precisely as described in the DGRC protocol [1]. Cl.8 cells recover slowly after thawing. Once they reach approximately 10^7 cells/ml (enough to cover the surface of the flask), they should be diluted as follows: Using a Pasteur pipet, blow medium at the surface of the flask to dislodge the cells; Cl.8 cells are the most strongly surface-adherent of the 4 modENCODE lines, but they can still be dislodged by this procedure. Transfer the contents of the flask to a 10 cm plate; add 5 ml of fresh medium to the plate and pipet gently up and down to mix. Add 5 ml of fresh medium to the flask; there are generally enough cells left on the surface that they will readily grow up. Sometimes, after a few days the cells in the plate do not seem as healthy as those in the flask; if this happens, go back to the flask and repeat the procedure. It will take several transfers for the cells to reach their normal growth rate; they should not be used for experiments during that time. General instructions for culture maintenance * Scale up cells as follows: Gently remove the cells from the substrate by blowing medium at the surface of the plate from a serological pipet. Cl.8 cells are more surface-adherent than the other 3 lines in the modENCODE collection, but they can still be dislodged in this way. Dilute the resulting cell suspension in fresh medium, and dispense to fresh tissue-culture grade Petri plates. Cl.8 cells are sensitive to over-dilution, and will not grow well at concentrations less than around 2-3x10^6 cells/ml. A culture should be scaled up when the cells cover the entire surface and are starting to pile up; at this point, they can be diluted approximately 5-fold. The line grows more slowly than Kc or S2; it will need to be transferred about once or twice a week. * For experiments, use them at mid-exponential growth, approximately the concentration shown in the photo on the DGRC web-page (https://dgrc.cgb.indiana.edu/files/repository/151.jpg?id=d5ccc5565da982039b9402ee90a92843) or a little higher. * Do not grow Cl.8 continuously for more than 4 months. This line is distinctly less stable than the other three lines. Go back to a frozen stock earlier if the cells look unhealthy.

value: dorsal mesothoracic disc


title: 1182-4H V026/BS23 (Celniker/RNA:39) rep1 extraction1_seq1 aliquote 1

source_name: 1182-4H cell small ncRNA RNA-Seq

value: embryo-derived cell-line

value: late embryonic stage

value: mh


title: adult female,eclosion + 1 day (Celniker/RNA:373) extraction1_seq1 aliquote 1

source_name: adult female,eclosion + 1 day (Celniker/RNA:373) extraction1_seq1 channel_1

growth_protocol:
Fly population cages contained the sequenced D. melanogaster isogenic strain with the mutations; yellow (y1), cinnabar (cn1), brown (bw1), and speck (sp1) [1]. Two homemade Plexiglas cages approximately 25cm x 25cm x 25cm, were used for staged embryo collections. These population cages were constructed from ¼ inch solid Plexiglas except for the open front panel. The fronts of the cages were covered with sleeves constructed of muslin cloth that were attached to the Plexiglas sides using Velcro strips. Each muslin sleeve was then twisted and held closed with a hose clamp. Flies were fed with three hard egg lay collection plates made in 150 X 15 mm Petri dishes containing a substrate of 3.3% agar, 13% unsulfured molasses, and 0.15% Tegasept. The hard egg lay plates were completely covered with a thin layer of moist yeast paste (Fleischmann's Baker's Dry Yeast) and placed horizontally on a short 1cm raised Plexiglas bar in the bottom of each cage to avoid crushing flies. The two population cages were maintained at 24 C in a controlled environment on a 24-hour light cycle (14 hours light / 10 hours dark) using three 60W bulbs on a timer. Flies were introduced to the cages at least four days prior to collections and fertility counts. These animals were initially grown in bottles at the same controlled environment described above. Each population cage was established from four trays with 35 bottles per tray containing newly eclosed adult flies. Each 250ml (pint) bottle contained approximately 40ml standard Drosophila medium in use at Bloomington (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/media-recipes.htm). The isogenic Drosophila mutant strain was propagated in bottles (A) starting with approximately 40-50 adults per bottle on day zero. New bottles (B) were generated from these bottles (A) on day 14. Cages were established by clearing the 'A' bottles into an empty bottle (without food) and transferring the newly eclosed adult flies in the 'A' bottles into the two population cages on day 18. The next round of cages and bottles (C) were established from the 'B' bottles in the same manner. Two-hour embryo collections were made during the light cycles following at least one two-hour pre-lay.
Protocol for staging adult flies for RNA isolation.

value: Adult female, eclosion + 1 day


title: Sg4 V011/BS35 (Celniker/RNA:201) rep2 extraction2_seq1 aliquote 1

source_name: Sg4 cell small ncRNA RNA-Seq


title: CME W1 Cl.8+ (Celniker/RNA:63) extraction1_seq1 aliquote 1

source_name: CME W1 Cl.8+ (Celniker/RNA:63) extraction1_seq1 channel_1

value: third instar larval stage


title: Celniker/RNA:9 extraction2_seq1 aliquote 1

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

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

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

value: Unknown


title: ML-DmD20-c5 (Celniker/RNA:187) extraction1_seq1 aliquote 1

source_name: ML-DmD20-c5 (Celniker/RNA:187) extraction1_seq1 channel_1

value: antenna disc-derived cell-line


title: Lai ML-DmD21 (Celniker/RNA:53, Replicate 2) extraction2_seq1 aliquote 1

source_name: ML-DmD21 (Celniker/RNA:53) extraction1_seq1 channel_1


title: adult male,eclosion + 1 day (Celniker/RNA:367) extraction1_seq1 aliquote 1

source_name: adult male,eclosion + 1 day (Celniker/RNA:367) extraction1_seq1 channel_1

value: Adult male, eclosion + 1 day


title: Tissue:Adult_ovaries:EL:1 extraction1_seq1 aliquote 1_Ago1IP_2747


title: Nuc_Mock_treated

city: Boston

first: Karen

last: Adelman

state: MA

address: 45 Shattuck St. LHRRB-201a

department: Biological Chemistry and Molecular Pharmacology

country: USA

institute: Harvard Medical School

description: MNase-digested_chromatin_mononucleosomal_fragments

library_source: genomic

data_processing: Paired-end reads were mapped with Bowtie 0.12.3 to the D. melanogaster, Flybase, r5.22 index (Langmead et al., 2009).  Biological replicates of untreated samples were in good agreement and were combined, resulting in a data set of 28,494,410 unique read pairs identifying both ends of fragments >=120 bp and =<180 bp in length.

molecule: genomic DNA

growth_protocol: S2(DGRC) cells were grown in M3 media (Sigma) supplemented with bacto-peptone and yeast extract + 10% FBS (GIBCO). S2 cells(Invitrogen) were grown in Schneider's media (GIBCO) supplemented with 10% FBS (GIBCO).

extract_protocol: MNase-digested chromatin from untreated, Mock-treated and NELF-depleted S2 cells was prepared as described in (Gilchrist et. al.,Genes Dev. 2008 Jul 15;22(14):1921-33) except that 200 µl chromatin was digested with 20 units MNase (Worthington) for 45 minutes at 25 degrees C. Following gel purification, mono-nucleosome sized fragments (100-200 bp) were subjected to paired-end sequencing using the Illumina paired-end protocol.

treatment_protocol: For RNAi experiments, S2 cells (DGRC) were harvested  96 hours after addition of dsRNA targeting NELF (NELF-B and NELF-E subunits; NELF-depleted) or  β-galactosidase (Mock-treated) as described in Armknecht, S. et al. (2005) Methods in Enzymology, 392: pp. 55-73.

tag: cell type

value: S2 cells


title: Nuc_untreated_1


title: Tissue:Adult_ovaries:EL:1 extraction1_seq1 aliquote 1_Ago1IP_2746


title: input DNA

status: Public on Oct 14 2010

city: New York

first: Yu

last: Chen

state: NY

address: 1275 York Ave, Box 20

department: Human Oncology and Pathogenesis Program

institute: Memorial Sloan Kettering Cancer Center

description: input DNA

data_processing: All reads (~36-bp) from ChIP-Seq were aligned to the human genome (build 36, or hg18) using the ELAND alignment software within the Illumina Analysis Pipeline. Unique reads mapped to a single best-matching location with no more than two mismatches were kept and used to generate genome-wide distribution of ETV1-binding and for peak identification. Redundant reads were considered once.

organism: Homo sapiens

source_name: GIST48 Cells, input

growth_protocol: GIST48 cells were continued passaged. 1E8 cells were used.

extract_protocol: Crosslinking ChIP was performed as described (Lee et al. 2006). Briefly, GIST cells were harvested and crosslinked in 1% paraformaldehyde for 15 minutes, pellets flash-frozen in liquid nitrogen, and stored at -80 degrees C.  Chromatin was isolated and sheared to 200-700 bp using a Bioruptor (Diagenode), and ChIP was performed as described (Lee et al. 2006).  Sheared chromatin was incubated with anti-rabbit IgG dynabeads pre-conjugated with anti-ETV1 antibody (Abcam Ab81086, Lot 787879), washed, eluted, reverse cross-linked, and purified. Purified ChIP DNA was blunt-ended, ligated to Solexa adaptors, amplified with 18-cycles of PCR. Following isolation of ChIP DNA, libraries were prepared for cluster generation as described (Goldberg et al. 2010).  Purified DNA libraries were used for cluster generation on Illumina/Solexa flow cells, and sequencing analysis was performed on an Illumina/Solexa Genome Analyzer 2 following manufacturer protocols.

value: GIST

tag: fraction

value: input control

tag: chip antibody


title: S2-DRSC V008/BS11 (Celniker/RNA:15) rep2 extraction2_seq1 aliquote 1

source_name: S2-DRSC cell small ncRNA RNA-Seq

value: Male


title: ETV1-ChIPSeq

description: Chromatin IP against ETV1

source_name: GIST48 Cells, ChIP fraction

value: ChIP fraction

value: anti-ETV1 (Abcam Ab81086, lot#787879)


title: CSHL_RnaSeq_GM12878_nucleus_TAP-Only_shortTotal

status: Public on Oct 08 2010

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

institute: ENCODE DCC

molecule: nuclear RNA

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

tag: datatype

value: RnaSeq

tag: datatype description

value: Sequencing analysis of RNA expression

tag: cell description

value: B-lymphocyte, lymphoblastoid, International HapMap Project - CEPH/Utah - European Caucasion, Epstein-Barr Virus

tag: rnaextract

value: shortTotal

tag: rnaextract description

value: Rna shorter than 200 nt that has not been seperated based on Poly Adenalyation

tag: biorep

tag: labexpid

tag: labversion

value: Produced by Hannon Lab,iIDR

tag: readtype

tag: readtype description

value: Single 36 nt reads

tag: replicate

tag: localization description

value: Large membrane bound part of cell containing chromosomes and the bulk of the cell's DNA

tag: protocol

value: TAP-Only

tag: protocol description

value: This kind of library was made from RNA < 200 nucleotides that were pre-treated with Tobacco Acid Pyrophosphatase to remove any 5' caps that would preclude cloning. Hence, both capped and 5' monophosphate RNAs will be cloned.


title: CSHL_RnaSeq_K562_nucleus_TAP-Only_shortTotal

tag: localization

value: nucleus


title: SssI MBDCap-Elu5

city: Zuerich

first: Mark

last: Robinson

address: Winterthurerstrasse 190

department: Institute of Molecular Life Sciences

country: Switzerland

institute: University of Zuerich

description:
library strategy: MBDCap-Seq
library selection: MBDCap

data_processing:
Bowtie was used mapping, allowing 3 mismatches. Only reads mapping to unique genomic locations were kept. For the comparison to array data, read counts were generated using annotationCounts() in the Repitools package (Statham et al. 2010 Bioinformatics) and statistics for differential methylation were calculated using the edgeR package (Robinson et al. 2010 Bioinformatics).
The count tables from the sequencing data that were used to generate the comparative results to the array outputs are available on the Series record (TSS_counts.txt).
For comparison of array and sequencing data, the outputs of blocksStats() -- a procedure used for targeted analysis of differential methylation: blocksStats_diffMeth.txt supplementary file is provided on the Series record.

source_name: SssI MBDCap-Elu5

growth_protocol: 37 deg C with 5% CO2

extract_protocol:
Sequencing libraries were prepared using the Chip-Seq DNA Sample Prep Kit (Illumina) according to the manufacturer's protocol
MBDCap: The MethylMiner™ Methylated DNA Enrichment Kit (Invitrogen) was used to isolate methylated DNA. 1 µg of genomic DNA was sonicated to 100-500 bps. 3.5 µg (7 µl) of MBD-Biotin Protein were coupled to 10 µl of Dynabeads M-280 Streptavidin according to the manufacturer’s instructions. The MBD-magnetic beads conjugates were washed 3 times and resuspended in 1 volume of 1 x Bind/Wash buffer. The capture reaction was performed by adding 1 µg sonicated DNA to the MBD-magnetic beads on a rotating mixer for 1 hour at room temperature. All capture reactions were done in duplicate. The beads were washed three times with 1 x Bind/Wash buffer. The methylated DNA was eluted in one of two ways: (1) as a single fraction with a High Salt Elution Buffer (2000mM NaCl), denoted MBD-SF, or (2) as distinct subpopulations based on the degree of methylation using an increasing NaCl concentration of the elution buffer, from 200mM to 2000mM in a stepwise gradient (Elution 1- 200mM, Elution 2 – 350mM, Elution 3 – 450mM, Elution 4 -600mM, Elution 5 -1000mM and Elution 6 – 2000mM). Each fraction was concentrated by ethanol precipitation using 1 µl glycogen (20 µg/µl), 1/10th volume of 3M sodium acetate, pH 5.2 and two sample volumes of 100% ethanol and resuspended in 60 µl H20.

tag: cell type

tag: mbdcap


title: CSHL_RnaSeq_K562_chromatin_TAP-Only_shortTotal

tag: datatype

value: chromatin

value: Nuclear DNA and associated proteins


title: embryos,2-6 hr after egg laying (Celniker/RNA:217, 223) extraction1-2

description:
embryos,2-4 hr after egg laying (Celniker/RNA:217) extraction1_seq1 aliquote 1
embryos,4-6 hr after egg laying (Celniker/RNA:223) extraction2_seq1 aliquote 1

extract_protocol:
Protocol for isolation of insect RNA using Trizol.  This protocol is used to prepare RNA that will be used for the isolation of microRNAs.  [TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml)]
mRNAs were purified from total RNA extracts, using standard Illumina protocol.  Briefly, 10ug total RNA was bound to oligo(dT) beads, washed twice, and eluted with 10mM Tris-HCl, using Ambion kit.
A Gel purification of RNA sample1. Spike 50 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.

tag: strain

value: Y cn bw sp

tag: developmental stage

value: Embryo 2-4h, 4-6h (mixture)

tag: genotype


title: SssI MBDCap-SF (lane 1)

source_name: SssI MBDCap-SF

value: SF


title: ML-DmBG1-c1 V075/BS176 (Celniker/RNA:115) rep1 extraction1_seq1 aliquote 1

source_name: ML-DmBG1-c1 cell small ncRNA RNA-Seq

tag: cell line

tag: tissue

value: CNS-derived cell-line

value: third instar larval stage

value: y v f mal

tag: Sex

value: Unknown


title: PrEC MBDCap-SF (lane 2)

source_name: PrEC MBDCap-SF

value: PrEC


title: PrEC MBDCap-SF (lane 1)


title: SssI MBDCap-SF (lane 2)


title: CSHL_RnaSeq_K562_cell_TAP-Only_shortTotal

value: immortalized cell line

tag: line

value: RnaSeq

tag: datatype description

value: Sequencing analysis of RNA expression

tag: rnaextract

value: shortTotal

tag: rnaextract description

value: Rna shorter than 200 nt that has not been seperated based on Poly Adenalyation

tag: biorep

tag: labexpid

tag: labversion

value: cell

value: Whole cell


title: CSHL_RnaSeq_K562_cytosol_TAP-Only_shortTotal

molecule: cytoplasmic RNA

value: Produced by Hannon Lab,iIDR

tag: readtype

tag: readtype description

value: Single 36 nt reads

tag: replicate

tag: localization

value: cytosol

tag: localization description

value: The fluid between the cells outer membrane and the nucleus

tag: protocol

value: TAP-Only

tag: protocol description

value: This kind of library was made from RNA < 200 nucleotides that were pre-treated with Tobacco Acid Pyrophosphatase to remove any 5' caps that would preclude cloning. Hence, both capped and 5' monophosphate RNAs will be cloned.


title: LNCaP MBDCap-SF (lane 2)

source_name: LNCaP MBDCap-SF

tag: cell type

value: LNCaP

tag: mbdcap

value: SF


title: LNCaP MBDCap-SF (lane 1)


title: Nuc_NELF_depleted

value: S2 cells


title: Reference Epigenome: ChIP-Seq Input from hESC H1 Cells; renlab.Input.hESC-06.01

status: Public on Oct 07 2010

city: La Jolla

first: UCSD AND

last: SALK

address: Health Sciences Drive

institute: University of California, San Diego

description:
sample_term_id: EFO_0003042
assay_term_id: OBI_0000716
nucleic_acid_term_id: SO_0000352
Design description: ChIP-Seq Input from hESC H1 Cells. Sequencing was done on the Illumina GAII platform.
Library name: DM219
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.3723
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.3673
****************
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: GSM605338_UCSD.H1.Input.DM219.bed
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.Input.hESC-06.01.hg19.level.1
ANALYSIS TITLE: Mapping of H1 Cell Line ChIP-Seq Input Data
ANALYSIS DESCRIPTION: Illumina reads produced by ChIP-Seq Input on the H1 Cell Line, Library DM219 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.4413
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: ChIP-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: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
ALIGNMENT_POSTPROCESSING: None
READ_EXTENSION: 200bp
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_Input_EXPERIMENTS_SCORED: 67
FINDPEAKS_SCORE: 0.1111
FINDPEAKS_PERCENTILE: 91
HOTSPOT_SCORE: 0.1569
HOTSPOT_PERCENTILE: 83
IROC_SCORE: 0.8251
IROC_PERCENTILE: 52
POISSON_SCORE: 0.3142
POISSON_PERCENTILE: 78

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

ANALYSIS FILE NAME: GSM605338_UCSD.H1.Input.DM219.wig
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.Input.hESC-06.01.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of H1 Cell Line ChIP-Seq Input Data
ANALYSIS DESCRIPTION: Illumina ChIP-Seq Input read mappings from the H1 Cell Line, Library DM219 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.4547
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: ChIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 200bp
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: H1 Input 19
BROWSER_TRACK_DESCRIPTION: UCSD H1 Cell Line ChIP-Seq Input Library DM219 EA Release 2

QUALITY SCORES:
NUMBER_OF_Input_EXPERIMENTS_SCORED: 67
FINDPEAKS_SCORE: 0.1111
FINDPEAKS_PERCENTILE: 91
HOTSPOT_SCORE: 0.1569
HOTSPOT_PERCENTILE: 83
IROC_SCORE: 0.8251
IROC_PERCENTILE: 52
POISSON_SCORE: 0.3142
POISSON_PERCENTILE: 78

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

source_name: Human embryonic stem cells; H1 cell line; renlab.Input.hESC-06.01

extract_protocol: Library construction protocol: See http://bioinformatics-renlab.ucsd.edu/RenLabLibraryProtocolV1.pdf

tag: molecule

value: genomic DNA

tag: disease

value: James Thompson Laboratory

value: Cell Line

tag: lineage

value: Embryonic Stem Cell

tag: passage

tag: medium

value: mTeSER

tag: Sex

value: Male

tag: batch

value: ChIP-Seq Input

value: Biorupter

value: Input

value: 500 micrograms


title: Reference Epigenome: ChIP-Seq Analysis of H4K5ac in hESC Cells; renlab.H4K5ac.hESC-01.01

description:
sample_term_id: EFO_0003042
assay_term_id: OBI_0000716
nucleic_acid_term_id: SO_0000352
Design description: ChIP-Seq Analysis of H4K5ac in hESC Cells. Sequencing was done on the Illumina GAII platform.
Library name: LL323
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.3669
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.1595
****************
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: GSM605330_UCSD.H1.H4K5ac.LL323.bed
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.H4K5ac.hESC-01.01-renlab.H4K5ac.hESC-02.01.hg19.level.1
ANALYSIS TITLE: Mapping of H1 Cell Line Histone H4K5ac ChIP-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by Histone H4K5ac ChIP-Seq on the H1 Cell Line, Library LL323 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.5188
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: ChIP-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: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
ALIGNMENT_POSTPROCESSING: None
READ_EXTENSION: 200bp
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_H4K5ac_EXPERIMENTS_SCORED: 4
FINDPEAKS_SCORE: 0.2986
FINDPEAKS_PERCENTILE:
HOTSPOT_SCORE: 0.3499
HOTSPOT_PERCENTILE:
IROC_SCORE: 0.9758
IROC_PERCENTILE:
POISSON_SCORE: 0.4644
POISSON_PERCENTILE:

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

ANALYSIS FILE NAME: GSM605330_UCSD.H1.H4K5ac.LL323.wig
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.H4K5ac.hESC-01.01-renlab.H4K5ac.hESC-02.01.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of H1 Cell Line Histone H4K5ac ChIP-Seq Data
ANALYSIS DESCRIPTION: Illumina Histone H4K5ac ChIP-Seq read mappings from the H1 Cell Line, Library LL323 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.4465
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: ChIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 200bp
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: H1 H4K5ac 23
BROWSER_TRACK_DESCRIPTION: UCSD H1 Cell Line Histone H4K5ac Library LL323 EA Release 2

QUALITY SCORES:
NUMBER_OF_H4K5ac_EXPERIMENTS_SCORED: 4
FINDPEAKS_SCORE: 0.2986
FINDPEAKS_PERCENTILE:
HOTSPOT_SCORE: 0.3499
HOTSPOT_PERCENTILE:
IROC_SCORE: 0.9758
IROC_PERCENTILE:
POISSON_SCORE: 0.4644
POISSON_PERCENTILE:

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

source_name: Human embryonic stem cells received from the James Thompson laboratory.  Cells are from the H1 line.; renlab.H4K5ac.hESC-01.01

tag: line

value: Between 25 and 45

value: Histone H4K5ac

value: magnetic anti-rabbit

value: 5 micrograms

value: Upstate


title: CSHL_RnaSeq_GM12878_cytosol_TAP-Only_shortTotal

tag: datatype

value: RnaSeq

tag: datatype description

value: Sequencing analysis of RNA expression

tag: cell description

value: B-lymphocyte, lymphoblastoid, International HapMap Project - CEPH/Utah - European Caucasion, Epstein-Barr Virus

tag: rnaextract

value: shortTotal

tag: rnaextract description

value: Rna shorter than 200 nt that has not been seperated based on Poly Adenalyation

tag: biorep

tag: labexpid

tag: labversion

value: Produced by Hannon Lab,iIDR

tag: readtype

value: Single 36 nt reads

value: The fluid between the cells outer membrane and the nucleus

value: TAP-Only

value: This kind of library was made from RNA < 200 nucleotides that were pre-treated with Tobacco Acid Pyrophosphatase to remove any 5' caps that would preclude cloning. Hence, both capped and 5' monophosphate RNAs will be cloned.


title: Reference Epigenome: ChIP-Seq Analysis of H3K9me3 in hESC Cells; renlab.H3K9me3.hESC-03.01

description:
Design description: ChIP-Seq Analysis of H3K9me3 in hESC Cells. Sequencing was done on the Illumina GAII platform.
Library name: YL75
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.3671
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.3524
****************
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: GSM605327_UCSD.H1.H3K9me3.YL75.bed
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.H3K9me3.hESC-03.01.hg19.level.1
ANALYSIS TITLE: Mapping of H1 Cell Line Histone H3K9me3 ChIP-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by Histone H3K9me3 ChIP-Seq on the H1 Cell Line, Library YL75 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.4432
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: ChIP-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: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
ALIGNMENT_POSTPROCESSING: None
READ_EXTENSION: 200bp
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_H3K9me3_EXPERIMENTS_SCORED: 59
FINDPEAKS_SCORE: 0.1566
FINDPEAKS_PERCENTILE: 71
HOTSPOT_SCORE: 0.1788
HOTSPOT_PERCENTILE: 56
IROC_SCORE: 0.7452
IROC_PERCENTILE: 29
POISSON_SCORE: 0.319
POISSON_PERCENTILE: 27

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

ANALYSIS FILE NAME: GSM605327_UCSD.H1.H3K9me3.YL75.wig
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.H3K9me3.hESC-03.01.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of H1 Cell Line Histone H3K9me3 ChIP-Seq Data
ANALYSIS DESCRIPTION: Illumina Histone H3K9me3 ChIP-Seq read mappings from the H1 Cell Line, Library YL75 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.4544
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: ChIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 200bp
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: H1 H3K9me3 75
BROWSER_TRACK_DESCRIPTION: UCSD H1 Cell Line Histone H3K9me3 Library YL75 EA Release 2

QUALITY SCORES:
NUMBER_OF_H3K9me3_EXPERIMENTS_SCORED: 59
FINDPEAKS_SCORE: 0.1566
FINDPEAKS_PERCENTILE: 71
HOTSPOT_SCORE: 0.1788
HOTSPOT_PERCENTILE: 56
IROC_SCORE: 0.7452
IROC_PERCENTILE: 29
POISSON_SCORE: 0.319
POISSON_PERCENTILE: 27

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

source_name: Human embryonic stem cells received from the James Thompson laboratory.  Cells are from the H1 line.; renlab.H3K9me3.hESC-03.01

tag: molecule

value: genomic DNA

value: James Thompson Laboratory

value: Cell Line

value: Embryonic Stem Cell

value: Between 20 and 40

value: mTeSER

value: Male

value: Histone H3K9me3

value: Biorupter

value: Abcam


title: Reference Epigenome: ChIP-Seq Analysis of H3K9me3 in hESC Cells; renlab.H3K9me3.hESC-04.01

description:
sample_term_id: EFO_0003042
assay_term_id: OBI_0000716
nucleic_acid_term_id: SO_0000352
Design description: ChIP-Seq Analysis of H3K9me3 in hESC Cells. Sequencing was done on the Illumina GAII platform.
Library name: YL77
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.3676
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.3524
****************
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: GSM605328_UCSD.H1.H3K9me3.YL77.bed
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.H3K9me3.hESC-04.01.hg19.level.1
ANALYSIS TITLE: Mapping of H1 Cell Line Histone H3K9me3 ChIP-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by Histone H3K9me3 ChIP-Seq on the H1 Cell Line, Library YL77 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.4403
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: ChIP-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: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
ALIGNMENT_POSTPROCESSING: None
READ_EXTENSION: 200bp
RELEASE_NUMBER: Human Epigenome Atlas 2

QUALITY SCORES:
NUMBER_OF_H3K9me3_EXPERIMENTS_SCORED: 59
FINDPEAKS_SCORE: 0.1492
FINDPEAKS_PERCENTILE: 68
HOTSPOT_SCORE: 0.2029
HOTSPOT_PERCENTILE: 68
IROC_SCORE: 0.7939
IROC_PERCENTILE: 41
POISSON_SCORE: 0.3933
POISSON_PERCENTILE: 58

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

ANALYSIS FILE NAME: GSM605328_UCSD.H1.H3K9me3.YL77.wig
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: renlab.H3K9me3.hESC-04.01.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of H1 Cell Line Histone H3K9me3 ChIP-Seq Data
ANALYSIS DESCRIPTION: Illumina Histone H3K9me3 ChIP-Seq read mappings from the H1 Cell Line, Library YL77 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.4557
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: ChIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 200bp
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: If multiple reads map to the same start position on the + strand or stop position on the - strand, only a single read is retained.
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: H1 H3K9me3 77
BROWSER_TRACK_DESCRIPTION: UCSD H1 Cell Line Histone H3K9me3 Library YL77 EA Release 2

QUALITY SCORES:
NUMBER_OF_H3K9me3_EXPERIMENTS_SCORED: 59
FINDPEAKS_SCORE: 0.1492
FINDPEAKS_PERCENTILE: 68
HOTSPOT_SCORE: 0.2029
HOTSPOT_PERCENTILE: 68
IROC_SCORE: 0.7939
IROC_PERCENTILE: 41
POISSON_SCORE: 0.3933
POISSON_PERCENTILE: 58

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

source_name: Human embryonic stem cells received from the James Thompson laboratory.  Cells are from the H1 line.; renlab.H3K9me3.hESC-04.01

tag: disease

value: 500 micrograms

value: magnetic anti-rabbit

value: 5 micrograms