Journal: Nature protocols

Article Title: Simultaneous reprogramming and gene editing of human fibroblasts

doi: 10.1038/nprot.2018.007

Figure Lengend Snippet:

Article Snippet: It is recommended that the cells be checked to ensure that they are chromosomally stable and that they are not infected with mycoplasma. list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 DMEM, high glucose (Thermo Fisher Scientific, cat. no. 11995-073) Dulbecco’s PBS (DPBS; Thermo Fisher Scientific, cat. no. 14190-144) FBS (Interpath Services, cat. no. SFBSF) GlutaMAX Supplement (Thermo Fisher Scientific, cat. no. 35050-061) Non-essential amino acids (Thermo Fisher Scientific, cat. no. 11140-050) TrypLE Select (Thermo Fisher Scientific, cat. no. 12563-029) pCMV-tdTomato vector (Clontech, cat. no. 632534) Reprogramming list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Plasmids: pEP4 E02S ET2K (Addgene, plasmid ID 20927), pEP4 E02S EN2L (Addgene, plasmid ID 20922), pEP4 E02S EM2K (Addgene, plasmid ID 20923), and pSimple-miR302/367 (Addgene, plasmid ID 98748) TesR-E7 reprogramming medium (Stem Cell Technologies, cat. no. 05910) Sodium butyrate (Sigma-Aldrich, cat. no. B5887) In vitro transcription of EBNA1, Cas9, and Cas9-Gem mRNA list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Plasmids: pSP6-EBNA 2A + DBD (Addgene, plasmid ID 98749), pDNR-SpCas9-Gem (Addgene, plasmid ID 80424), and pDNR-SpCas9 (Addgene, plasmid ID 80425) PmeI restriction endonuclease (NEB, cat. no. R0560S) EcoRI-HF restriction endonuclease (NEB, cat. no. R3101S) mMessage mMachine SP6 Transcription Kit (Thermo Fisher Scientific, cat. no. AM1340) mMessage mMachine T7 Ultra Transcription Kit (Thermo Fisher Scientific, cat. no. AM1345) RNA ladder (Promega, cat. no. G3191) Pluripotent stem cell culture list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 hESC-qualified Matrigel (LDEV (lactose dehydrogenase-elevating virus)-free; Corning, cat. no. 354277) DMEM/F-12 (Thermo Fisher Scientific, cat. no. 11320-082) Essential 8 medium (Thermo Fisher Scientific, cat. no. A1517001) DMSO (Sigma-Aldrich, cat. no. D5879) Genotyping analysis list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Custom-designed ODNs to generate ~200- to 800-bp amplicons around the sgRNA target site and identify targeted clones (Integrated DNA Technologies; see Supplementary Table 1 for primer sequences used in our experiments) GoTaq Green Master Mix (Promega, cat. no. M7123) Exonuclease I (NEB, cat. no. M0293S) rAPid alkaline phosphatase (Sigma-Aldrich, cat. no. 4898133001) BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, cat. no. 4337455) DNeasy Blood & Tissue Kit (Qiagen, cat. no. 69506)

Techniques: Plasmid Preparation

Journal: PLoS ONE

Article Title: MicroRNA-Mediated In Vitro and In Vivo Direct Conversion of Astrocytes to Neuroblasts

doi: 10.1371/journal.pone.0127878

Figure Lengend Snippet: Transduction and continuous expression of vectors were confirmed by injecting a GFP expressing empty lentiviral vector and lentiviral particle that included the miR-302/367+GFP cluster. Scale bar: 100 μm.

Article Snippet: We prepared the miR-302/367 cluster as lentiviral particles which included a GFP expressing sequence (System Biosciences, San Francisco, CA) by transfecting along with a Virapower Lentiviral Packaging Mix (Invitrogen) into 293T cells by the Lipofectamine 2000 Transfection Reagent (Invitrogen).

Techniques: Transduction, Expressing, Plasmid Preparation