A retroviral sgRNA library was designed targeting all the sites bearing PAMs throughout a 20-kb region surrounding the Foxp3 locus (from 12.5 kb upstream to 7.5 kb downstream of the transcription start site). Specifically, we first identified all PAM sequences (NGG) within the X chromosome: 7143000–7165000 (mm9). To ensure the target specificity, we filtered the targets with low complexity sequences, such as repetitive ACG and TTTT. We then selected the targets ending with PAM and bearing unique sequence specificity in the mouse genome. In addition, 100 nontargeting negative controls were added to the library (Table S1 ). A library containing 75-nt single-stranded DNA oligonucleotides was synthesized by using a high-throughput method (GenScript) and amplified by PCR with primers binding to the flanking arms according to standard protocols. PCR product was then assembled with BbsI-linearized pSIR-BbsI-Thy1.1 vector backbone (modified from pSIR-hCD2, Cat51143; Addgene) by using Gibson Assembly Master Mix (New England Biolabs). The resulting product was electroporated into Escherichia coli (New England Biolabs) and selected by ampicillin on 16 25-cm2 dishes. Transformation efficiency was quantified to ensure sufficient coverage. Plasmid was extracted by using the CompactPrep Plasmid Maxi Kit (QIAGEN), and the sgRNA coverage was validated by performing high-throughput sequencing.
Escherichia coli
Manufactured by New England Biolabs
Sourced in United States
Escherichia coli (E. coli) is a common bacterium widely used in laboratory settings. It is a Gram-negative, rod-shaped organism that is a key model organism for scientific research. E. coli serves as a versatile tool for various applications, such as cloning, protein expression, and genetic engineering experiments.
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Lab products found in correlation
Gibson assembly master mix, by New England Biolabs (1 mentions)
Compactprep plasmid maxi kit, by Qiagen (1 mentions)
Albumin dextrose catalase, by BD (1 mentions)
Oleic acid albumin dextrose catalase, by BD (1 mentions)
Chelex 100 resin, by Bio-Rad (1 mentions)
Middlebrook 7h9 broth, by BD (1 mentions)
Penicillin streptomycin, by Quality Biological (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
4 protocols using escherichia coli
1
CRISPR-Based Screening of Foxp3 Locus
2
Mycobacterial Growth and Media Preparation
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All mycobacterial strains were grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) containing 0.2% glycerol, 0.1% Tween 80, and 10% albumin-dextrose-catalase (Becton, Dickinson and Co., Sparks, MD) with rotation at 37°C. Middlebrook 7H10 solid medium supplemented with 10% oleic acid-albumin-dextrose-catalase (Becton, Dickinson and Co., Sparks, MD) was used to isolate single colonies. The 7H9 and 7H10 media were supplemented with mycobactin J (mJ) (Allied Monitor, IN) as indicated. Iron-depleted glycerol-alanine salts with Tween 80 (GAST) medium was prepared using Chelex 100 resin (Bio-Rad) (10 g/liter) for the mycobactin production and radioactive iron uptake assays (8 (link)). Chemically competent Escherichia coli (New England BioLabs) was grown in Luria-Bertani medium (Difco) at 37°C. Antibiotics were added when needed as follows: apramycin at 60 μg/ml for E. coli ; hygromycin at 100 μg/ml for E. coli and 50 μg/ml for mycobacteria; and kanamycin at 50 μg/ml for both E. coli and mycobacteria. All chemicals used were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.
3
SARS-CoV-2 Viral Protein Cytopathic Effects in Fission Yeast
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A wild-type fission yeast SP223 strain (h-, ade6-216, leu1-32, and ura4-294) was used to test the cytopathic effect of SARS-CoV-2 viral proteins in this study (8 (link), 14 (link)). Standard yeast extract-sucrose (YES) complete, minimal EMM, or Pombe Glutamate medium (PMG) selective media supplemented with adenine, uracil, leucine, or thiamine (20 μM) was used to grow fission yeast cells or to select for plasmid-carrying cells. Luria-Bertani (LB) medium supplemented with ampicillin (100 μg/mL) was used for growing New England BioLabs (NEB) stable Escherichia coli (NEB catalog [cat] no. C3040H) or DH5α cells and for DNA transformation.
Human pulmonary epithelial cell lines A549 (ATCC CCL-185) and Calu-3 (ATCC HTB-55) and a human embryonic kidney epithelial 293T cell line was used in this study. A549 and 293T cell lines were maintained in the high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Corning cat 10-017-CV) with 10% fetal bovine serum (FBS; Gibco cat 100-438-026) and 100 U/mL penicillin-streptomycin (Gibco cat 15140122). Calu-3 cells were maintained in Eagle’s minimum essential medium (EMEM) (Quality Biological cat 112-018-101) with 10% FBS and 100 U/mL penicillin-streptomycin. All cell lines were grown in an incubator at 37°C with 5% CO2.
Human pulmonary epithelial cell lines A549 (ATCC CCL-185) and Calu-3 (ATCC HTB-55) and a human embryonic kidney epithelial 293T cell line was used in this study. A549 and 293T cell lines were maintained in the high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Corning cat 10-017-CV) with 10% fetal bovine serum (FBS; Gibco cat 100-438-026) and 100 U/mL penicillin-streptomycin (Gibco cat 15140122). Calu-3 cells were maintained in Eagle’s minimum essential medium (EMEM) (Quality Biological cat 112-018-101) with 10% FBS and 100 U/mL penicillin-streptomycin. All cell lines were grown in an incubator at 37°C with 5% CO2.
4
Microbial Diversity and Interactions
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Blautia coccoides (ATCC® 29236; ATCC, VA, USA), Escherichia coli (New England Biolabs, MA, USA) and Lactobacillus casei (Saga University) were used in this study.
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Blautia coccoides (ATCC® 29236; ATCC, VA, USA), Escherichia coli (New England Biolabs, MA, USA) and Lactobacillus casei (Saga University) were used in this study.
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