|
| Status |
Public on Aug 30, 2012 |
| Title |
ctrl_trik4_r2_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
wing imaginal discs, LID RNAi knock-down, input
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: LID RNAi
developmental stage: third instar larvae
tissue: wing imaginal discs
chip antibody: none
sequencing_date: Oct_2010
|
| Treatment protocol |
The LID RNAi strain was obtained from NIG-FLY and it was combined with lidk06801. For every sample, around 500 wing imaginal discs were dissected in PBS.
|
| Growth protocol |
w1118, act5Cgal4>LID RNAi or lidk06801;act5Cgal4>lid RNAi larvae were grown at 25ºC until they became wall-climbing third instar larvae.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Discs were crosslinked in 1.8% formaldehyde solution and sonicated. DNA-protein complexes were immunoprecipitated with anti-LID or anti-H3K4me3 antibodies. Commercial Abcam ab8580 antibodies were used as anti-H3K4me3. Custom-made anti-LID polyclonal antibodies were raised in rabbits from GST fusion proteins containing fragments of LID, and IgGs were purified for the ChIP experiments. Libraries were prepared according to Illumina's instructions accompanying the ChIP-Seq DNA Sample Prep Kit (Part# IP-102-1001). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters, which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with Illumina primers for 16 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 40 (H3K4me3) and 36 (LID) nucleotides on the Illumina Genome Analyzer II following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Input DNA from LID RNAi knock-down dm (in a mutant background).
|
| Data processing |
Alignment files: FASTQ files were obtained with the standard Illumina Pipeline version 1.5.1. Reads were aligned to the dm3 genome with Bowtie version 0.12.5, keeping sequences mapping to a unique location in the genome with up to 2 mismatches in the first 28 bases. We removed PCR over-amplification artifacts by only keeping the first 100 appearances of a given sequence for analysis. We removed strand-specific biases with a procedure analogous to that proposed for the MACS algorithm.
Peak files: We defined peaks using a two-step procedure. Step 1: We found enriched genomic regions showing high accumulation of sequences in the IP sample compared to the control. We selected regions with coverage above 10 and compared the proportion of reads inside/outside of each region between the IP sample and its corresponding control with a logistic regression likelihood-ratio test. We defined enriched regions as those with Benjanimi-Yekutieli adjusted P-value < 0.05. Step 2: We defined peaks as location within the enriched regions (determined in Step 1) with coverage above 50. Bioconductor software was used for all data analysis unless where otherwise specified.
|
| |
|
| Submission date |
Feb 04, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
David Rossell |
| E-mail(s) |
[email protected]
|
| Organization name |
IRB Barcelona
|
| Street address |
Baldiri Reixac 10
|
| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9061 |
| Series (2) |
| GSE27078 |
LID ChIP-Seq in wild type, and H3K4me3 ChIP-Seq in wild type and lid RNAi Drosophila melanogaster |
| GSE27081 |
Drosophila dKDM5/LID regulates H3K4me3 dynamics at the transcription start site of actively transcribed developmental genes |
|
| Relations |
| SRA |
SRX040616 |
| BioSample |
SAMN00205542 |
