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Comment on Version 2

The following citation to Bio-protocol has been added to the beginning of the “Glucosinolate and cyanogenic glucoside uptake assays” section in the Materials and methods:

Uptake assays in Xenopus laevis oocytes using liquid chromatography–mass spectrometry to detect transport activity is described in more detail at Bio-protocol (Jørgensen et al, 2017a).

A second citation to Bio-protocol has been added to the following sentence to the “Cloning of synthesized genes into Xenopus expression vector” section in the Materials and methods:

Design and direct assembly of synthesized uracil-containing non-clonal DNA fragments into vectors by USER cloning is described in more detail at Bio-protocol (Jørgensen et al, 2017b).

The two corresponding references have also been added to the article:

Jørgensen, ME, Crocoll, C, Halkier, BA, Nour-Eldin, HH. (2017a). Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity. Bio-protocol 7(20): e2581. DOI: https://doi.org/10.21769/BioProtoc.2581

Jørgensen, ME, Wulff, N, Nafisi, M, Xu, D, Wang, C, Lambertz, SK, Belew, ZM, Nour-Eldin, HH. (2017b). Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning. Bio-protocol 7(22): e2615. DOI: https://doi.org/10.21769/BioProtoc.2615

Comment on Version 2

The following citation to Bio-protocol was added to the following sentence in the Fluorescence-activated cell sorting (FACS) section of the Materials and Methods:

Tumors were dissociated into a single cell suspension according to protocol established in our lab (McCauley and Guasch, 2013) and CD34+ cancer stem cells were isolated as described at Bio-protocol (McCauley and Guasch, 2017).

The following reference has also been added to the Reference section:

McCauley, HA, Guasch G. (2017). Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma. Bio-protocol 7(17): e2524. DOI: https://doi.org/10.21769/BioProtoc.2524

Comment on Version 2

The following citation to Bio-protocol was added after the first sentence in the "Dynabead assay" section of the Materials and Methods:

The detailed protocol is available in Bio-protocol (Sawaguchi et al. 2017).

The following reference has also been added to the Reference section:

Sawaguchi, S, Ogawa, M, Okajima, T. (2017). Protocol for Notch-ligand Binding Assays Using Dynabeads. Bio-protocol 7(20): e2582. DOI: https://doi.org/10.21769/BioProtoc.2582

Note from eLife: Co-submitted with Brent W Bisgrove, Yi-Chu Su and H Joseph Yost, "Maternal Gdf3 is an obligatory cofactor in nodal signaling for embryonic axis formation in zebrafish", available at https://elifesciences.org/articles/28534 and Tessa Grace Montague and Alexander F Schier, "Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm", available at https://elifesciences.org/articles/28183.

Note from eLife: Co-submitted with Jose L Pelliccia, Granton A Jindal and Rebecca D Burdine, "Gdf3 is required for robust Nodal signaling during germ layer formation and left-right patterning", available at https://elifesciences.org/articles/28635, and Brent W Bisgrove, Yi-Chu Su and H Joseph Yost, "Maternal Gdf3 is an obligatory cofactor in nodal signaling for embryonic axis formation in zebrafish", available at https://elifesciences.org/articles/28534.

Note from eLife: Co-submitted with Tessa Grace Montague and Alexander F Schier, "Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm", available at https://elifesciences.org/articles/28183, and Jose L Pelliccia, Granton A Jindal and Rebecca D Burdine, "Gdf3 is required for robust Nodal signaling during germ layer formation and left-right patterning", available at https://elifesciences.org/articles/28635.

Comment on Version 2

The following sentence was added to the end of then Materials and methods subsection titled “Single-cell RNA-seq”.

The raw and processed RNA-seq data for the 288 cells can be found on the GEO database (accession number: GSE105054).

The dataset was cited in the Data sets section:

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

Sumin Jang Vilas Menon Anne-Rachel Krostag Boaz P Levi Sharad Ramanathan (2017)

Publicly available at the NCBI Gene Expression Omnibus (accession no. GSE105054).

Comment on Version 2

The following citation to Bio-protocol was added to the start of the Materials and Methods section titled "Stereotaxic adeno-associated virus injection and cannula implantation":

The surgery procedure is described in more detail at Bio-protocol (Correia et al., 2017).

The following reference has also been added to the Reference section:

Correia, PA, Matias, S, Mainen, ZF. (2017). Stereotaxic Adeno-associated Virus Injection and Cannula Implantation in the Dorsal Raphe Nucleus of Mice. Bio-protocol 7(18): e2549. DOI: https://doi.org/10.21769/BioProtoc.2549

Comment on Version 2

The following citation to Bio-protocol was added to the start of the Materials and Methods section titled "Immunohistochemistry and cell counting":

Memory trace labeling and analyses are described in more detail at Bio-protocol (Du and Koffman, 2017).

The following reference has also been added to the Reference section:

Du, J, Koffman, EE. (2017). Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System. Bio-protocol 7(20): e2578. DOI: https://doi.org/10.21769/BioProtoc.2578

Comment on Version 4

An incorrect, previous version of the 240-well WorMotel mold STL file was inadvertently published, this has now been replaced with the correct version of the mold that was used in the experiments. The previous mold had a 1 mm thick base and wall. When the PDMS is demolded using a spatula, there is considerable stress on the mold that sometimes caused it to crack. The new mold design has a thicker 2 mm base and wall and is resistant to cracking.