 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 11, 2014 |
| Title |
PFR40_EEmb_HPL2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
HPL-2 ChIP DNA for PFR40 EEmb
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: PFR40
developmental stage: Early Embryo
genotype: hpl-2(tm1489)
chip antibody: HPL-2 SDI Q2324
Sex: population predominantly Hermaphrodites perhaps with some Males
|
| Growth protocol |
Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Embryos were then frozen in liquid nitrogen.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_popcorn_chromatin_extract_protocol_vSS1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in a Diagenode Bioruptor (40 pulses of 30 seconds with 1 minute rests in between at full power). Extracts are then spun down and the soluble fraction is stored for quality tests and future ChIP. Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 μl of protein A dynabeads (Dyna), and washed 5 minutes with 1 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 μL elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Qiagen PCR purification columns.
|
| Label |
Cy5
|
| Label protocol |
ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays Users Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.
|
| |
|
| Channel 2 |
| Source name |
Input DNA for PFR40 EEmb
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: PFR40
developmental stage: Early Embryo
genotype: hpl-2(tm1489)
sample type: input DNA
Sex: population predominantly Hermaphrodites perhaps with some Males
|
| Growth protocol |
Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Embryos were then frozen in liquid nitrogen.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_popcorn_chromatin_extract_protocol_vSS1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in a Diagenode Bioruptor (40 pulses of 30 seconds with 1 minute rests in between at full power). Extracts are then spun down and the soluble fraction is stored for quality tests and future ChIP. Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 μl of protein A dynabeads (Dyna), and washed 5 minutes with 1 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 μL elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Qiagen PCR purification columns.
|
| Label |
Cy3
|
| Label protocol |
ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays Users Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.
|
| |
|
| |
| Hybridization protocol |
ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.
|
| Scan protocol |
ChIP-chip_scanning_nimblegen_v1. Array scanning and raw data extraction were performed at Roche NimbleGen, according to the protocol described in chapter 5 and 6 of the NimbleGen Arrays Users Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, array signal was scanned by using a GenePix 4000B Scanner with associated software and saved as .tif files of the 532nm and 635nm images individually. Raw signal intensities of the images were extracted and saved as .pair files by using NimbleScan software according to the NimbleScan v2.4 User?s Guide.
|
| Description |
ChIP-chip of HPL-2 in PFR40 early embryos
|
| Data processing |
The log2-ratio ratio of intensity from sample/reference channel was standardized to obtain z-scores. Signal was lifted from ce4 to ce10 using the liftOver tool found at the UCSC Genome Browser website (http://hgdownload.cse.ucsc.edu/admin/exe/).
|
| |
|
| Submission date |
Jun 24, 2014 |
| Last update date |
Oct 11, 2014 |
| Contact name |
Susan Strome |
| E-mail(s) |
sstrome@ucsc.edu
|
| Phone |
831-459-4615
|
| Organization name |
UC Santa Cruz
|
| Department |
Molecular, Cell, and Developmental Biology
|
| Lab |
329 Sinsheimer Labs
|
| Street address |
1156 High St.
|
| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95064 |
| Country |
USA |
| |
|
| Platform ID |
GPL8647 |
| Series (2) |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1419169_PFR40_EEmb_HPL2_IP_OID29066_19217901_635.pair.gz |
36.2 Mb |
(ftp)(http) |
PAIR |
| GSM1419169_PFR40_EEmb_HPL2_Inp_OID29066_19217901_532.pair.gz |
36.2 Mb |
(ftp)(http) |
PAIR |
| GSM1419169_PFR40_EEmb_HPL2_z_ce10.wig.gz |
8.6 Mb |
(ftp)(http) |
WIG |
| Processed data included within Sample table |
|
|
|
|
 |
