Geneticin selective antibiotic g418 sulfate

An S100A11 short hairpin (sh)RNA vector was designed and synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China), with a target sequence of 5′-GGATGGTTATAACTACACT-3′. A scramble shRNA vector was used as a negative control. HO8910 cells were transfected with S100A11 or control shRNA using Lipofectamine^® LTX with Plus^™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The stable cell clones were isolated using Geneticin^® Selective Antibiotic (G418 Sulfate; Gibco Life Technologies, Beijing, China).

Bevacizumab was obtained from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Human immunoglobulin G (HuIgG) was purchased from MP Biomedicals (Santa Ana, CA, USA). Both Bevacizumab and HuIgG were diluted with normal saline. Na-pyruvate and Geneticin™ Selective Antibiotic (G418 Sulfate) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). RPMI-1640 culture medium, d-glucose, and HEPES buffer were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was obtained from Bovogen Biologicals Pty. Ltd. (Melbourne, Australia).

For the generation of the auxin-inducible degradation system for DPY30 sgRNA targeting the stop-codon region were cloned into eSpCas9(1.1)-T2A-eGFP. Left and right homology arms, as well as the mAID-T2A-BFP middle part were ligated into a modified pUC19 vector backbone (a gift from S. Pollard) using the In-Fusion cloning kit (Takara, 638910). mES cells were co-transfected with sgRNA- and donor-vector using Lipofectamine 3000 and sorted 48 h later for GFP/BFP-double-positive cells. Homozygous clones were then transfected with pPB-hygro-OsTIR1-P2A-mCherry and pBase plasmids and selected with 100 µg ml⁻¹ hygromycin B (Thermo Fisher Scientific, 10687010). For the generation of the endogenous dTAG-inducible degradation system for RBBP5, sgRNA targeting the stop codon region were co-transfected into the cells and contained the following elements: left and right homology arms, as well as FKBP12(F36V), 2× HA tags, P2A and a neomycin-resistance gene. The transfected cells were selected with 100 µg ml⁻¹ Geneticin selective antibiotic (G418 Sulfate) (Thermo Fisher Scientific, 10131027), single-cell sorted to obtain clonal cell lines and screened for correct biallelic integration. All homozygous insertions and knock-ins were confirmed by Sanger sequencing and western blotting. A list of the oligos and the sequences of the sgRNAs is provided in Supplementary Table 1.

The SARS-CoV-2 USA_WA1/2020 (SARS-CoV-2/human/USA/WA-CDC-02982586-001/2020, GenBank: MN985325.1) and B.1.1.529 (Omicron) sublineage BA.5.5 (hCoV-19/USA/COR-22-063113/2022, GISAID accession ID: EPI_ISL_13512579) were obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA, University of Texas Medical Branch, Galveston, TX, USA). Both viruses were passaged once in VeroE6-TMPRSS2 cells for subsequent experiments and deep sequenced to verify single nucleotide variants (SNVs) frequencies along the whole genome at the UTMB’s Next Generation Sequencing (NGS) Core, directed by Dr. Steven G. Widen. Deep sequencing of these stocks revealed no mutations or deletions in the Spike protein >5.0% frequency.
VeroE6 cell line expressing human TMPRSS2 were obtained from JCRB Cell Bank (JCRB1819, lot 04172020)^{36 (link)}. VeroE6-TMPRSS2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 100 µg/ml of Geneticin™ Selective Antibiotic - G418 Sulfate (Thermo Fisher Scientific). VeroE6-TMPRSS2 cell line was verified and tested negative for mycoplasma before use.

500 mL DMEM/F-12 (Thermo Fisher, cat. no. 11320033)
(If cell line can be selected by Geneticin add 4 mL Geneticin™ selective Antibiotic (G418 Sulfate) (50 mg/mL) (Thermo Fisher, cat. no. 10131027))
50 mL Fetal Bovine Serum, certified and heat inactivated (Thermo Fisher, cat. no. 10082139 or US 10082147)
5.5 mL Penicillin-Streptomycin (10,000 U/mL) (Thermo Fisher, cat. no. 15140122)

For the generation of the auxin-inducible degradation system for DPY30 sgRNA targeting the stop-codon region were cloned into eSpCas9(1.1)-T2A-eGFP. Left and right homology arms, as well as the mAID-T2A-BFP middle part were ligated into a modified pUC19 vector backbone (a gift from S. Pollard) using the In-Fusion cloning kit (Takara, 638910). mES cells were co-transfected with sgRNA- and donor-vector using Lipofectamine 3000 and sorted 48 h later for GFP/BFP-double-positive cells. Homozygous clones were then transfected with pPB-hygro-OsTIR1-P2A-mCherry and pBase plasmids and selected with 100 µg ml⁻¹ hygromycin B (Thermo Fisher Scientific, 10687010). For the generation of the endogenous dTAG-inducible degradation system for RBBP5, sgRNA targeting the stop codon region were co-transfected into the cells and contained the following elements: left and right homology arms, as well as FKBP12(F36V), 2× HA tags, P2A and a neomycin-resistance gene. The transfected cells were selected with 100 µg ml⁻¹ Geneticin selective antibiotic (G418 Sulfate) (Thermo Fisher Scientific, 10131027), single-cell sorted to obtain clonal cell lines and screened for correct biallelic integration. All homozygous insertions and knock-ins were confirmed by Sanger sequencing and western blotting. A list of the oligos and the sequences of the sgRNAs is provided in Supplementary Table 1.