GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1541735

Query DataSets for GSM1541735

Status

Public on Jan 14, 2015

Title

USV101 tagged capsule non-inducing mock

Sample type

SRA

Source name

cultured cells

Organism

Cryptococcus neoformans

Characteristics

strain: KN99alpha
genotype: USV101-HA-tagged
chip antibody: rabbit IgG anti-HA antibody
condition description: capsule non-inducing
condition: 30°C + YPD

Treatment protocol

Cells were transfered to grow for 90 minutes in either capsule-inducing (DMEM, 37 °C, 5% CO2) or capsule non-inducing (YPD, 30 °C) conditions

Growth protocol

Wild-type and HA-tagged strains were cultured overnight in YPD to a density of 1-2 OD600.

Extracted molecule

genomic DNA

Extraction protocol

The cells were fixed with formaldehyde, lysed by mechanical bead-beating, and the cell debris was removed by centrifugation. The supernatant fraction, containing isolated chromatin, was sheared by sonication, cleared again by centrifugation, and an aliquot was reserved as ‘input’. The remaining material was incubated with rabbit IgG anti-HA antibody (Abcam) tethered to protein A sepharose (‘IP’) or sepharose alone (‘mock’) overnight at 4°C. The beads were then washed, incubated at 65°C to reverse DNA-DNA and DNA-protein crosslinks, and the DNA recovered by phenol/chloroform/isoamyl alcohol (25:24:1) extraction, ethanol precipitation, and resuspension in nuclease-free water.
ChIP-DNA for for all samples was end-repaired with Klenow DNA Polymerase and the DNA was purified with AMPure XP System beads (Beckman Coulter Genomics) and modified with A-tails using Klenow exo- before ligation to adapters to incorporate 7-base index sequences using T4 DNA ligase (Enzymatics). Adapter addition was confirmed on an Agilent 2100 bioanalyzer, and the DNA was PCR-amplified and then gel purified to remove adapter dimers and select sizes optimal for high-throughput sequencing (150 to 300 bp). Samples were submitted to the Washington University Genome Technology Access Center for library preparation and multiplex sequencing.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

ChIP-seq mock control: USV101 tagged capsule non-inducing

Data processing

Sequenced reads were aligned to the C. neoformans H99 reference sequence (v2) using Bowtie version 0.12.8
Peak calling was performed with MACS (v1.4.0) by comparing tagged IP reads as the treatment sample and input, mock, and untagged IP reads as the control samples.
For NRG1 analysis, tagged IP treatment replicates 2 & 3 were compared with the untagged wildtype IP control. Any gene falling completely or partially within 1,000 bp on either side of a peak’s summit was considered ChIP positive for that IP-control pair. Only genes that were ChIP-positive for both IP-control pairs were considered NRG1 bound. For Usv101, ChIP-positive genes were called separately for cells grown in capsule-inducing and non-inducing conditions. In each condition, the tagged IP treatment was compared the tagged input and mock controls, and the untagged wildtype IP control. Any gene falling completely or partially within 1,000 bp on either side of a peak’s summit was considered ChIP positive for that IP-control pair. Only genes that were ChIP-positive for all three IP-control pairs were considered USV101 bound.
Genome_build: C. neoformans H99 reference sequence (Cryptococcus neoformans var. grubii H99 Sequencing Project)
Supplementary_files_format_and_content: bed (peaks): Peak calling was performed with MACS (v1.4.0). Each line a peaks bed file gives the coordinates and score (-10log10(pval)) of a MACS identified peak.

Submission date

Nov 09, 2014

Last update date

May 15, 2019

Contact name

Ezekiel John Maier

E-mail(s)

[email protected]

Organization name

Washington University

Department

Computer Science