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| Status |
Public on Aug 29, 2020 |
| Title |
Klac_WT_IP1_2 |
| Sample type |
SRA |
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| Source name |
Log-phase culture in BMW
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| Organism |
Kluyveromyces lactis |
| Characteristics |
genotype: wild type
replicate: 2
chip antibody: αSfp1-1 custom polyclonal antibody (Bethyl)
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| Treatment protocol |
NA
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| Growth protocol |
Log-phase culture in rich media
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The ChIP protocol was performed as described by Lefrancois et al. in The Guide to Yeast Genetics (Weissman, Jonathan F., Guthrie, 2010). Polyclonal antibodies were created (Bethyl, Inc) to bind a peptide from the conserved c-terminal portion of Sfp1 (NH2-Cys-GKRYKNLNGLKYHRGHSTH-COOH), designated, αSfp1-1, while αSfp1-3 was raised to recognize was raised to bind specifically to the N. castellii Sfp1pl (using NH2-Cys-QLKKNSTASSSHQNSNNQTH-COOH). In all cases, the antibody used for ChIP was αSfp1-1, with the exception of those N. castellii IP3 samples, which used αSfp1-3.
DNA was quantified using Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). Input samples were diluted to the same concentration of ChIP samples. End Repair was carried out with the End-It DNA repair Kit (Epicenter). An AMPure Spri XP bead (Agencourt) clean-up was performed after end repair. A-base addition was carried out with 5 ul of NEB reagents Klenow Buffer (NEB Buffer #2), 10 ul of 1mM dATP (NEB) and 1 ul of Klenow exo (3’ to 5’ exo -; NEB) to each sample. An AMPure Spri XP bead clean-up was performed after A-base addition. Adaptor ligation was completed using 15 ul of 2x Quick DNA ligase buffer (NEB),1 ul of custom indexed adapters at uM, and 1 ul of Quick T4 DNA ligase (NEB) to each sample. An AMPure Spri XP bead clean-up was performed after adapter ligation. PCR was carried out using 1 ul of the PFU enzyme (Agilent), 5 ul of Pfu Buffer (Agilent), 0.5 ul of 100 uM dNTPs, and 2.5 ul of nuclease free water. An AMPure Spri XP bead clean up and samples were eluted in 15 ul of 10 mM Tris-Cl, pH 8.5. Samples were pooled in an equal molar ratio prior to sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Reads were mapped to their respective genomes using Bowtie2
Convert SAM files to big wig read coverage files
calculate the ratio of each ChIP to input signal (both antiSfp1-ChIP in WT and antiSfp1-ChIP in deltaSfp1)
Calculate the log ratio of these ratios (ChIP_WT/input_WT)/(ChIP_dSfp1/input_dSfp1).
Extract the ratios for various genomic elements (promoters, gene bodies); identify those that are significantly bound compared to the background.
Genome_build: S288C R64 for S. cerevisiae, YGOB v7 Aug 2012 for K. lactis and N. castellii, and Scannell et al. for S. paradoxus (Scannell et al., 2011)
Supplementary_files_format_and_content: Processed data files include both raw signal tracks (.unique.bw), log ratios relative to both input and to sfp1 mutants.
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| Submission date |
Aug 29, 2018 |
| Last update date |
Aug 30, 2020 |
| Contact name |
Carl G de Boer |
| Organization name |
The Broad Institute
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| Lab |
Aviv Regev
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| Street address |
415 Main St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL19759 |
| Series (2) |
| GSE119211 |
SFP1 (ChIP-seq) |
| GSE119238 |
The roles of duplication and divergence in the evolution of a transcription factor |
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| Relations |
| BioSample |
SAMN09934228 |
| SRA |
SRX4623266 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3360770_Klac_WT_IP1_2.unique.bw |
31.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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