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Sample GSM8279340

Query DataSets for GSM8279340

Status

Public on May 26, 2024

Title

plasmid DNA fragments, amplified, library after sorting in the second round

Sample type

SRA

Source name

Escherichia coli

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

cell type: Escherichia coli

Growth protocol

The transformed cells were incubated in LB broth (1:4, v/v) for 1.5 h at 37 °C to recover and washed with fresh M9 medium with the corresponding antibiotic (1:1, v/v) once. The resulting culture was incubated with 100 ml of modified M9 medium (with chloramphenicol and ampicillin) in 500 ml flasks with shaking at 30℃ and 250 rpm. The cultures were induced with 1 mM IPTG at an OD600 of about 1.0 and allowed to grow for an additional 40 h to sample the cell library (before sorting, BS). The cell library (before sorting, BS) was dyed with Nile red and sorted by FACS, obtaining the cell library after sorting (AS).

Extracted molecule

other

Extraction protocol

Plasmid extraction was performed using the TIANprep Mini Plasmid Kit (Tiangen, China). The plasmids were used as templates for PCR to amplify the N20 region of library sgRNAs. The PCR fragment was purified using the TIANquick Midi purification Kit (Tiangen, China).
For each sample, the purified PCR products were treated with End Prep Enzyme Mix for end repairing, 5’ phosphorylation, and dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Adaptor-ligated DNA was then performed using DNA Clean Beads. A second PCR reaction was carried out with P5 and P7 primers carrying sequences which can anneal with flowcell to perform bridge PCR and index allowing for multiplexing. The products were purified by beads and then qualified. The final libraries were sequenced via the Illumina NovaSeq 6000 platform using a 2 × 150 paired-end configuration.

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

NGS-second_(B-cul2_B-sor3-P5).xlsx

Data processing

The original data of sequencing results were subjected to image Base calling by using CASAVA 1.8.2, and the quality was preliminarily analyzed, getting the Pass Filter Data. The sequencing data was optimized by using Cutadapt 1.9.1, removing the primers and adapter, the bases with the quality score below 20 at both ends, and the reads with N base ratio more than 10%. Clean Reads are merged to generate a complete sequence by using Pandaseq 2.7 according to the overlap regions between Read1 and Read2. The target sequence is captured according to the 10 nt of upstream and downstream of the target sequence. The merged sequences are aligned to the reference sequence by using BWA software, the section on the alignment is extracted, and abundance information is counted.
Assembly: GCF_000005845.2_ASM584v2
Supplementary files format and content: Excel file-the reads of sgRNAs in the first round of screening
Supplementary files format and content: Excel file-the reads of sgRNAs in the second round of screening
Library strategy: Amplicon sequencing

Submission date

May 19, 2024

Last update date

May 26, 2024

Contact name

Lixia Fang

E-mail(s)

lxfang@tju.edu.cn

Organization name

Tianjin University

Street address

135 Yaguan Road, Jinnan District