|
| Status |
Public on Nov 26, 2021 |
| Title |
Hot_concat_before_2 |
| Sample type |
SRA |
| |
|
| Source name |
S. pombe modified by genome engineering
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Strain CBS 2777 derived
modification: chromsome 2 centromere modified
chip target and antibody: CENP_A modified by amino terminal GFP tag and precipitated using Abcam Ab 290
|
| Treatment protocol |
see supplementary data
|
| Growth protocol |
Growth in YES to OD 0.7 at 600nm
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Material was quantified using Qubit (Invitrogen) and the size profile analysed on the 2200 or 4200 TapeStation (Agilent, dsDNA HS Assay). Input material was normalised to 5 ng or maximum available prior to library preparation. Automated library preparation was performed using the Apollo prep system (Wafergen, PrepX ILMN 32i, 96 sample kit) and standard Illumina multiplexing adapters following manufacturer’s protocol up to pre-PCR amplification. Libraries were PCR amplified (18 cycles) on a Tetrad (Bio-Rad) using the NEBNext High-Fidelity 2X PCR Master Mix (NEB) and in-house unique dual indexing primers (based on DOI: 10.1186/1472-6750-13-104). Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to ~10 nM for storage. The 10 nM library was denatured and further diluted prior to loading on the sequencer. Paired end sequencing was performed using a HiSeq4000 75bp platform (Illumina, HiSeq 3000/4000 PE Cluster Kit and 150 cycle SBS Kit)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Generated by CHIP~seq
FASTA file:
Not_76_Chr_2_Hot_region_concatamer_before_combined.fasta
|
| Data processing |
fastq files were not quality trimmed but aligned to customized fasta files (inlcluded) using the Burroughs Wheeler aligner in samtools. Bam files were filtered for paired sequences using the 99, 147, 83 and 163 flags and then merged into asingle bam file
Generation of the coverage vectors used in the figures used a Granges object using the R libraries, GenomicRanges, GenomicAlignments, rtracklayer, Rsamtools, limma,
Coverage vectors were normalized on the basis of read counts to the centromeres of chromosomes 1 and 3. Read counts were calculated using the samtools view command.
Coverage vectors were normalized on the basis of read counts to the centromeres of chromosomes 1 and 3. Read counts were calculated using the samtools view command.
Vectors were smoothed using a median smoothing ; using the runmed command on a vectorized derivative of the Rle object with a smoothing distance of either 101 or 1001 residues for either the centromere of whole chromsome coverage vectors
Genome_build: Not relevant used custom fasta files; FASTA files available on the series record.
Supplementary_files_format_and_content: coverage vectors that can be processed and inspected with simple text editors
|
| |
|
| Submission date |
Aug 10, 2021 |
| Last update date |
Nov 26, 2021 |
| Contact name |
WILLIAM RA BROWN |
| E-mail(s) |
william.brown@nottingham.ac.uk
|
| Phone |
01158230386
|
| Organization name |
Nottingham University
|
| Department |
Life Sciences
|
| Lab |
D9, QMC
|
| Street address |
Life Sciences Department, Queens Medical Centre
|
| City |
Nottingham |
| State/province |
NOTTS |
| ZIP/Postal code |
NG7 2UH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL22682 |
| Series (1) |
| GSE181806 |
Mutation and selection explain why eukaryotic centromeric DNA is often A+T rich |
|
| Relations |
| BioSample |
SAMN20696032 |
| SRA |
SRX11708813 |
