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Sample GSM4026878

Query DataSets for GSM4026878

Status

Public on Sep 30, 2019

Title

Combinatorial_library_plasmid_rep1

Sample type

SRA

Source name

Synthetic sequence

Organism

Escherichia coli

Characteristics

cell line: Top10

Treatment protocol

Cells harboring variant libraries, prepared as described above, were grown in the presence of 2 μg/mL doxycycline, incuding expression of the variant library. Cells were passaged over approximately two weeks, with a subset of cells collected and frozen for genomic extraction and sequencing after each replicate was complete.

Growth protocol

HEK 293T LLP-rEF1alpha cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin

Extracted molecule

other

Extraction protocol

Cells were transferred into a microfuge tube, pelleted and stored at -20˚C. Genomic DNA was prepared using the GentraPrep kit (Qiagen) or Dneasy kit (Qiagen).
40 ng of plasmid DNA was mixed with 0.333 µM of forward primer, 0.333 µM of reverse primer, and 2x Kapa Hifi added as half the volume (Supplementary table 1). The mixtures were cycled 12 times in a thermocycler. 1 µl of DPNI enzyme (20 units) was added to each tube, and incubated at 37◦C for 2 hours. 15 µl of product was mixed with 3 µl of (6x gel loading dye), and run on a 0.8 percent agarose gel in TBE. Bands were cut, position 1 bands and position 2 bands were pooled separately, and extracted using the Qiagen Gel Extraction kit. Position 1 and position 2 DNA were eluted in 10 µl nuclease-free water. 5 µl of each eluate were mixed together with 10 µl 2x Gibson mix and incubated at 50◦C for 30 minutes. After incubation, the product was eluted in 5 µl, and 4 µl of library was electroporated into 10-beta cells (NEB) using 0.1 µm cuvettes (Bio-Rad) according to manufacturer instructions. Approximately 2.16 million colony forming units of transformants were grown in 200 ml Luria Broth overnight, and plasmid DNA was extracted with a Qiagen Midiprep Kit (Qiagen). 320 ng of library DNA was mixed with 2x Kapa Hifi Readymix and a final concentration of 0.333 µM KAM2311 and 0.333 µM KAM2312, with 30 µl distributed into 8 replicate tubes. 160 ng of rIP2M_attB_GFP plasmid was mixed with 2x Kapa Hifi and a final concentration of 0.333 µM KAM2313 and 0.333 µM KAM2314, with 30 µl distributed into 4 replicate tubes. Both sets of tubes were amplified over 18 cycles. 1 µl (20 units) of DPNI enzyme was added to each tube, and incubated at 37◦C for 2 hours. After incubation, the rIP2M_attB_GFP amplicon was gel extracted from a 0.8% agarose gel and eluted in 10 µl, whereas the library amplicon was cleaned and concentrated (Zymo), and eluted in 5 µl. 4 µl of each were mixed together with 8 µl of 2x Gibson mix and incubated at 50◦C for 30 minutes. The product was clean-and-concentrated, eluted in 5 µl, and electroporated in 10-beta cells using 0.1 µm cuvettes (Bio-Rad) according to manufacturer's instructions. Approximately 0.5 million colony forming units were amplified in 200 ml Luria Broth overnight, and extracted using the Qiagen Midiprep kit. Sanger sequencing of 8 colonies revealed 6 to be intended library members. This library was called attB-TSPCR.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

Library and barcode cassette sequencing from DNA
Combination_indices.csv
Plasmid

Data processing

Libraries were sequenced on NextSeq 500 instruments (Illumina) and base called using the instruments' Real Time Analysis software.
Sequencing reads were converted to fastq format and de-multiplexed with bcl2fastq. R1 and R2 were trimmed to the first 16 nucleotides,which includes both the eight nucleotides of known sequence serving as a transgene identifier, as well as the flanking four degenerate nucleotides serving as unique plasmid identifier sequences. These datafiles were used to identify each unique plasmid molecule which had been recombined. In subsequent analyses only looking at the eight nucleotides serving as a transgene identifier, the initial fastq files were trimmed such that only nucleotides 5-12 remained. Both steps were performed using custom python scripts which are posted on the lab Github page.
The properly trimmed fastq files were concatenated to create a single sequence denoting the paired transgene combination. The resulting fastq file was entered into Enrich2, and Log2 enrichment scores for each transgene combination was calculated for each replicate experiment. Scores from replicate experiments were averaged to obtain a single average enrichment score, as well as its standard error.
Genome_build: NONE
Supplementary_files_format_and_content: The "Combination_indices.csv" files lists the indices that denote which transgene is present based on the Illumina sequencing data. The "Enrichment_scores.csv" file is the final result of running the Illumina fastq files through trimming (described above) for the indices, Enrich, and associating the enrichment scores for each index combination back to the transgenes they represent. In this file, the number following "e" represents the replicate number. Columns with the letter "s" following the number are columns containing the enrichment score for that replicate. Columns with the letter "e" contain the standard error for that replicate.

Submission date

Aug 12, 2019

Last update date

Sep 30, 2019

Contact name

Kenneth Matreyek

E-mail(s)

Kenneth.Matreyek@Case.edu

Organization name

Case Western Reserve University

Department

Pathology