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Sample GSM6398434

Query DataSets for GSM6398434

Status

Public on Sep 06, 2022

Title

BAT-CoV_N_RNA-SELEX_cycle3_rep4

Sample type

SRA

Source name

Rosetta (DE3)

Organism

Escherichia coli

Characteristics

tissue: Rosetta (DE3)
cell type: Rosetta (DE3)
genotype: WT
treatment: pET28+BAT-CoV_N vector transfected

Treatment protocol

1 mM IPTG (Isopropyl β-D-1-Thiogalactopyranoside) was added into the bacterial culture to induce protein expression at 16 °C for 16 h.

Growth protocol

The E. coli strains for protein purification were grown at 37 ℃ in LB (Luria-Bertani) broth with shaking at 220 rpm or on LB agar plates until OD600=0.6.

Extracted molecule

protein

Extraction protocol

The pellet was suspended in 10 ml buffer A (0.5 mg/ml Lysozyme, 50 mM tris-HCl pH 7.5, 300 mM NaCl, 10 mM Imidazole, 1 mM DTT, 1 mM PMSF) and lysed by sonication at ten seconds interval for 10 minutes and then centrifuged at 4 °C (12000 rpm, 30 min) to collect protein supernatant. The supernatant was filtered with a 0.45 μm filter and the filtrate was added into a Ni-NTA column (Bio-Rad) which had been equilibrated with buffer A before using. The Ni-NTA beads was set at 4 ℃ for 30min before being eluted with Buffer A containing 300 mM imidazole
HT-SELEX was performed by using selection ligands containing a 10 bp barcode after the 40 bp randomized region. The ligand libraries were produced by PCR amplification using the primers in Supplementary Table1 and 40 bp randomized oligo as template. The libraries were sequenced using BGI MGISEQ-2000 sequencer to analyze the uniformity of each base (A, T, C, G). RNA input was synthesized from the DNA-templates using T7 in vitro transcription and the remaining DNA template was digest by DNaseⅠ. To enable the RNA to form secondary structures, the RNA input libraries were then heated to 70°C followed by slow cooling. Then, 600 ng protein and 5 ul RNA input selection ligands were added into binding buffer (50 mM NaCl, 1 mM MgCl2, 0.5 mM Na2EDTA , 1U/μL RNase inhibitor and 4% glycerol in 50 mM Tris-Cl, pH 7.5) until the total volume reached 25 ul. After incubated for 15 minutes at 37°C, the mixture was followed by additional 15 minutes at room temperature. Subsequently, 150- µL binding buffer, containing 10 µL pre-equilibrated Ni Sepharose 6 Fast Flow resin (GE Healthcare, 17-5318-01) was added into the mixture. After incubated for further 2 hours with gentle shaking at room temperature, the beads were then washed with gentle shaking 12 times with 200 μL of binding buffer to remove the unbound RNA ligands. Subsequently, the residual moisture on beads was carefully cleared by a soft centrifuging at 500 g for 30 s, and then the bound RNA was resuspended in 20 μL elution buffer (0.5 µM RT-primer, 1 mM EDTA and 0.1% Tween 20 in 10 mM Tris-Cl buffer, pH 7.0) and heated for 5 minutes at 70°C followed by 4°C to anneal the reverse transcription primer to the remaining RNA. Finally, the eluted RNA was used for reverse transcription and PCR amplification using the the primers in Supplementary Table 1, and the obtained PCR products were used as DNA template for the next cycle after containing a T7 promoter. This process was repeated four times. Each PCR products were purified and sequence using BGI MGISEQ 2000 sequencer.

Library strategy

SELEX

Library source

other

Library selection

other

Instrument model

BGISEQ-500

Description

PCR products from BAT-CoV_N_RNA-SELEX_cycle3_rep4

Data processing

Illumina Casava v1.7 software used for basecalling.
Quality control of the raw sequencing data using FastQC version v0.11.7 with default parameters.
Sequences from the 40-nt random region without bases annotated as N were extracted using custom script.
Autoseed software was used for motif analysis.
The R ggseqlogog package was used to construct motif logos.
Assembly: NA
Supplementary files format and content: xls file was a tab-delimited file include the summary of position weight matrix(PWM) for all motifs.

Submission date

Jul 26, 2022

Last update date

Sep 06, 2022

Contact name

Jian Yan

Organization name

City University of Hong Kong

Department

Department of Biomedical Sciences

Lab

Yan Lab