GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1835642

Query DataSets for GSM1835642

Status

Public on Jan 18, 2016

Title

7-CBF-666774-uninduced

Sample type

SRA

Source name

CRISPRi guide RNA library_CBF-666774-uninduced

Organism

Saccharomyces cerevisiae

Characteristics

grna libraries: gene_tiling_18bp & gene_tiling_20bp
growth condition: 166.7uM CBF-666774
index pair: CGTTGA-AGGTC
molecule subtype: plasmid DNA

Treatment protocol

In general, drugs were applied at concentrations that inhibit growth by ~20%

Growth protocol

Yeast culturing and sample collection was performed using a cell-screening platform that integrates temperature-controlled absorbance plate readers, plate coolers, and a liquid handling robot. Briefly, 700 µl yeast cultures were grown (+/- a drug and +/- ATc) in 48 well plates at 30oC with orbital shaking in Infinite plate readers (Tecan). To maintain cultures in log phase over many doublings, 23 µls of the culture was removed when it reached an OD of 0.76, added to a well containing 700 µl of media, and then allowed to grow further. After three such dilutions, 600 µls of the culture was collected and saved to a 4oC cooling station (Torrey Pines) when it reached an OD of 0.76. This amounted to approximately 20 culture doublings from the beginning of the experiment. Pipetting events were triggered automatically by Pegasus Software and performed by a Freedom EVO workstation (Tecan).

Extracted molecule

genomic DNA

Extraction protocol

After competitive growth of yeast strains and sample collection, yeast plasmids were purified using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research).
Purified plasmids were used as a template for PCR with barcoded up and down sequencing primers that produce a double index to uniquely identify each sample. PCR products were confirmed by agarose gel electrophoresis. After PCR, samples were combined and bead cleaned with Thermo ScientificTM Sera-Mag Speed Beads Carboxylate-Modified particles. Sequencing was performed using Illumina MiSeq.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

Sample.7

Data processing

library strategy: Barcode sequencing (Bar-seq)
created a synthetic reference chromosome sequence for each of the expected amplicons. The synthetic reference included Illumina adaptors, library barcode, PCR amplification priming region, PCR barcode, and the gRNA specificity sequence.
merged forward and reverse reads using PEAR version 0.9.4 with default parameters.
resulting FastQ file was mapped against the created synthetic reference using BWA version 0.6.1-r104 with command line “bwa index [reference]; bwa aln -n3 -o3 -e1 -l22 [reference] [fastq] > [aln]; bwa samse -f [out] [reference] [aln] [fastq] ”
For each of the expected amplicons, we counted the number of perfect matches (flag NM:i:0) from the resulting alignments (Additional data file 8, column “Count_perfect”) that were used in subsequent analyses.
Supplementary_files_format_and_content: Additional data file 8. An Excel spreadsheet that contains sequence read information and raw counts from MiSeq data.

Submission date

Jul 29, 2015

Last update date

May 15, 2019

Contact name

Ulrich Schlecht

E-mail(s)

schlecht.ulrich@gmail.com

Phone

6502136281

Organization name

Stanford University