|
| Status |
Public on Jun 07, 2018 |
| Title |
ChIPSeq_pSpt5_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
spt5-13myc::kanMX6 leu1-32 ura4-D18 his3-D1 ade6-M210 h+
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
temperature c: 30
drug (5 minutes): None
antibody: Anti-pSpt5 antibody (custom made from 21st Century Biochemicals, Marlboro, MA; Sanso et al. 2012)
assay: ChIP-seq
|
| Treatment protocol |
For PRO-seq, Cells were spun down and resuspended in fresh YES medium, pre-conditioned at the desired temperature (30°C or 18°C) and allowed to incubate for 10 min. Either 10 uM 3-MB-PP1 or equivalent volume of DMSO was then added to samples for 5 minutes before harvesting.
|
| Growth protocol |
For PRO-seq, S. pombe cultures were grown in YES media from a starting OD600 of 0.2 to a final OD600 = 0.5 before treatment.
For ChIP-seq: S. pombe cultures were grown in YES media till OD600 ~ 0.4.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
PRO-seq: Immediately prior to treatments, a fixed amount of spike-in culture (i.e. S. cerevisaie samples) was added to each sample culture for PRO-seq experiment. After treatment, cultures were then spun down and washed once in ice-cold water. Samples were then spun again and resuspended in ice-cold permeabilization buffer (0.05% sarkosyl) and left on ice for 20 minutes. Permeabilized cells were then spun down at 400XG for 5 minutes. Permeabilized cell pellets were resuspeneded in 120 uL 2.5X transcription buffer (50 mM Tris-HCl, pH 7.7, 500 mM KCl, 12.5 mM MgCl2). To the reaction buffer we then added 3.75 uL of each biotin-11-NTP (epicentre), 6 uL .1M DTT, 3 uL SUPERase Inhibitor (invitrogen) and 141 uL DEPC treated water. The run-on was then performed by adding 15 uL 10% sarkosyl and placing samples at 30°C for 5 minutes. After the run-on, samples were spun and the reaction buffer was removed. RNA was then isolated using the hot acid phenol extraction protocol followed by ethanol precipitation.
ChIP-seq: After HCHO-crosslinking (15 min at 25°C) protein extract was made using Mini-beadbeater (Biospec; 30 sec "ON" and 30 sec "OFF"). Lysates were precleared with IgG and used for IP with respective antibodies.
PRO-seq libraries were prepared according to Mahat et al. Nature Protocols (2016).
ChIP-seq: Multiplexed ChIP-seq libraries were prepared using the Illumina TruSeq DNA Sample Preparation kit v2 with 75 ng of input or IP DNA and barcode adaptors.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
PRO-seq: Adaptor sequences were clipped from read 3' ends. In cases where inline barcodes were used, they were clipped from 5' ends. Sequences were trimmed to a maximum length of 36 (minimum = 15). The reverse complement of reads was generated for alignment.
PRO-seq: Using Bowtie (version 1.0) Reads were first aligned to ribosomal DNA genomes of both S. cerevisiae and S. pombe. Any reads that failed to align to ribosomal DNA were then aligned to a combinded genome of S. cerevisiae and S. pombe. We allowed for a maximum of 2 mismatches Only uniquely aligning reads were used for downstream analysis.
PRO-seq: Unique reads were parsed based on their species of origin. Bedgraph files were created by recording only the most 3' base of each read (which represents the position of the Pol II active site). The counts at each position in a bedgraph file were normalized as counts per million mappable spike-in reads. Normalized bedgraphs were then converted to bigwig formated files.
ChIP-seq: Paired-end sequencing (50-nt reads) was performed on an Illumina HiSeq 2000 (Genome Quebec Innovation Centre, McGill University).
ChIP-seq: After adaptor trimming and quality control, processed FASTQ files were aligned to the S. pombe genome using Bowtie2 (Galaxy Version 2.2.6.2).
ChIP-seq: Aligned sequences of each biological replicate were fed into MACS2 (Galaxy Version 2.1.1.20160309.0) to call peaks from alignment results. Generated “bedgraph treatment” files were concatenated (Galaxy Version 1.0.1) to combine replicates of each sample, converted into bigwig using “Wig/BedGraph-to-bigWig converter” (Galaxy Version 1.1.0) and processed using computeMatrix (Galaxy Version 2.3.6.0) in DeepTools to prepare data for plotting a heatmap and/ or a profile of given regions.
Genome_build: PRO-seq: S. pombe: ASM294v2; S. cerevisiae: S288C_reference_genome_R64-1-1_20110203.
Genome_build: ChIP-seq: S. pombe: ASM294v2.
Supplementary_files_format_and_content: PRO-seq: processed files are in bigwig format. There are two bigwig files for each sample, one for each strand. Each bigwig file contains normalized counts of read 3'-ends.
Supplementary_files_format_and_content: ChIP-seq: processed files are in bedraph format. There are 3 bedgraph files for each sample, two for biological replicates and one combined.
|
| |
|
| Submission date |
Aug 11, 2017 |
| Last update date |
Jun 07, 2018 |
| Contact name |
Robert P Fisher |
| E-mail(s) |
robert.fisher@mssm.edu
|
| Phone |
2126598677
|
| Organization name |
Icahn School of Medicine at Mount Sinai
|
| Department |
Oncological Sciences
|
| Lab |
15-76
|
| Street address |
One Gustave L. Levy Place, Box 1130, L15-76
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL13988 |
| Series (1) |
| GSE102590 |
A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II |
|
| Relations |
| BioSample |
SAMN07501014 |
| SRA |
SRX3090264 |
