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Sample GSM5224486

Query DataSets for GSM5224486

Status

Public on May 31, 2021

Title

CCM3_Ammo_30

Sample type

SRA

Source name

Synthetic sequence

Organism

Homo sapiens

Characteristics

cell line: HEK293T-LP
treatment: AmmocidinA_30nM
library: CCM
molecule subtype: PCR from gDNA

Treatment protocol

After selection with Blasticidin/Rimiducid, cells were transferred to 6 well plates for drug selection with ammocidin A or apoptolidin A added as a 1000x stock in DMSO. Cells were treated for four days and allowed to recover for three days, in two cycles. After treatment, cells were expanded, and genomic DNA extracted as below.

Growth protocol

HEK293 cells were maintained in glucose free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin, 10 mM galactose. After addition of the attB plasmid library, media was supplemented with 1 μg/mL doxycycline and successfully recombined cells were selected with 100 μg/mL Blasticidin S, 10 nM Rimiducid for 7 days.

Extracted molecule

genomic DNA

Extraction protocol

Confluent cells from 6 well dishes were scraped from the plate, pelleted at 800 g for 5 min, and genomic DNA was extracted using the Qiagen DNAeasy kit per manufactures protocols, and eluted in 100 μL of low EDTA TE buffer. The transgene region was amplified using primers targeted to the tetracycline promoter (located in the attP landing pad) and the IRES site (located in the attB plasmid), such that only successful integrated transgenes were amplified. PCR was carried out at 100 μL scale using NEB Q5 high fidelity polymerase per manufacturers protocols using 5 μL of genomic DNA, an annealing temperature of 68 °C, and a 3 min extension time to limit bias towards shorter products (ATP5C) for a total of 30 cycles. PCR amplicons were purified using the NEB Monarch PCR clean-up kit per manufactures protocols.
Nextera Flex library prepration on PCR amplicons

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

processed data file: gatk_counts_H293.csv

Data processing

library strategy: Targeted amplifaction
Adapter trimming and QC with fastp v 0.20.0
Alignment using bwa-mem 0.7.17 w/ -O 100
Generate bam/sam using samtools v1.10
Count variants using gatk v.4.1.8.1 AnalyzeSaturationMutagenesis
Collate data and tidy using R 4.0.4
Genome_build: NA
Supplementary_files_format_and_content: gatk_counts_library.csv - variant frequency in the library as a csv, chain = gene, aa.residue = WT residue/position, aa = amino acid, residue = position, count = num of counts of that variant observed in the data, clen = chain length, frac = fraction of library composed of the variant
Supplementary_files_format_and_content: gatk_counts_H293.csv - variant frequency recovered from cells as a csv, sample = replicate, compound = compound and dose, chain = gene, aa.residue = WT residue/position, aa = amino acid, residue = position, count = num of counts of that variant observed in the data, clen = chain length, frac = fraction of library composed of the variant

Submission date

Apr 01, 2021

Last update date

May 31, 2021

Contact name

Benjamin Reisman

E-mail(s)

[email protected]

Organization name

Vanderbilt University

Department

Chemistry