2i lif media  (Biosynth Carbosynth)

Journal: eLife

Article Title: Large-scale analysis of the integration of enhancer-enhancer signals by promoters

doi: 10.7554/eLife.91994

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , N27 , Gibco , #17504-044 , 2i+LIF media.

Techniques: Recombinant, Plasmid Preparation, Reporter Assay, Sequencing, RNA Extraction, Reverse Transcription, Ligation, Magnetic Beads, Software

Journal: bioRxiv

Article Title: Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity

doi: 10.1101/2023.05.06.539708

Figure Lengend Snippet: ( a ) Schematic describing four different versions of Dyad-seq. M-M-Dyad-seq profiles 5mC on one strand and if the cytosine on the opposing strand of the dyad is methylated or not. M-H-Dyad-seq profiles 5mC on one strand and if the cytosine on the opposing strand of the dyad is hydroxymethylated or not. H-H-Dyad-seq profiles 5hmC on one strand and if the cytosine on the opposing strand of the dyad is hydroxymethylated or not. H-M-Dyad-seq profiles 5hmC on one strand and if the cytosine on the opposing strand of the dyad is methylated or not. ( b ) 5mCpG maintenance, quantified as the percentage of CpG dinucleotides that are symmetrically methylated, is shown for mESCs grown under different conditions. M-M-Dyad-seq is used to estimate 5mCpG maintenance. ( c ) M-H-Dyad-seq shows the percentage of 5mC that are paired with 5hmC at CpG dyads. ( d ) H-H-Dyad-seq shows the percentage of 5hmC that are paired with 5hmC at CpG dyads. ( e ) H-M-Dyad-seq shows the percentage of 5hmC that are paired with 5mC at CpG dyads. ( f ) Genome-wide 5mCpG levels quantified using M-M-Dyad-seq. ( g ) Genome-wide 5hmCpG levels quantified using M-H-Dyad-seq. In panels (b,f), the violin plots are made using 100 kb bins, and in panels (c-e,g), the violin plots are made using 1 Mb bins. In panels (b-g), mESCs are grown under SL conditions before being transitioned to the following conditions for 48 hours: No – basal media, BL – basal media with LIF, SL – serum containing media with LIF, GL – basal media with LIF and GSK3i, 2iL – basal media with LIF, GSK3i and MEKi, or ML – basal media with LIF and MEKi. ( h ) Heatmap of differentially expressed genes with a putative role in regulating DNMT1-mediated maintenance fidelity.

Article Snippet: Commercial 2i media containing LIF (Millipore, SF016–200) was used for BL, GL, 2iL, and ML experiments.

Techniques: Methylation, Genome Wide

Journal: BMC Biology

Article Title: Ino80 is essential for proximal-distal axis asymmetry in part by regulating Bmp4 expression

doi: 10.1186/s12915-016-0238-5

Figure Lengend Snippet: Ino80 KO ESCs are viable, but differentiate when maintained in a metastable pluripotent state. a Microscopic analysis of wild-type and Ino80 KO pre-implantation E3.5 blastocysts. Blastocysts were allowed to outgrow for seven days onto gelatinized plates in media containing serum + LIF. Wild-type and Ino80 KO are at identical magnification. Red arrows designate ESC colony outgrowth. b Western analysis of Ino80 protein expression in wild-type and Ino80 KO ESCs using a custom Ino80 antibody. Ponceau S was used as a loading control. c Microscope analysis of wild-type and Ino80 KO ESCs grown in media containing serum + 2i + LIF or serum + LIF. Wild-type and Ino80 KO are at identical magnification. d Alkaline phosphatase (AP) activity in wild-type and Ino80 KO ESCs after plating at clonal density and maintained in media containing serum + 2i + LIF or serum + LIF. Wild-type and Ino80 KO are at identical magnification. e Percent AP positive colonies quantified from panel d and after Ino80 KO ESCs passaged in serum + LIF were recovered in serum + 2i + LIF. At least 50 colonies were quantified per group. f Quantitative RT-PCR analysis of gene expression in wild-type and Ino80 KO ESCs maintained in serum + 2i + LIF. (N = 3 biological replicates) g Quantitative RT-PCR analysis of gene expression in wild-type and Ino80 KO ESCs maintained in serum + LIF. ( N = 3 biological replicates; * = t test p ≤ 0.05). KO knockout, ESC embryonic stem cells, E embryonic day, LIF leukemia inhibitory factor,

Article Snippet: Expanded blastocysts were hatched using Tyrodes solution (Sigma T1788) and allowed to attach and outgrow on gelatinized plates for seven days in defined 2i + LIF media without serum (Millipore Cat# SF016-100).

Techniques: Western Blot, Expressing, Control, Microscopy, Activity Assay, Quantitative RT-PCR, Gene Expression, Knock-Out

Journal: BMC Biology

Article Title: Ino80 is essential for proximal-distal axis asymmetry in part by regulating Bmp4 expression

doi: 10.1186/s12915-016-0238-5

Figure Lengend Snippet: Ino80 localizes to Bmp4 promoter and regulates its expression during embryonic stem cell differentiation. a Quantification of Bmp4 , Hex , Lefty1 , Cer1 , Gata6, and Gata4 expression by RT-qPCR during ESC differentiation in serum containing media without 2i + LIF in monolayer (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). b Cartoon showing the Bmp4 gene, location of upstream enhancers, and immediate downstream convergent gene Gm15217 position of PCR amplicons used in chromatin immunoprecipitation (ChIP) experiments are shown as black bars labeled 1-6. c Localization of Ino80 to regulatory elements of Bmp4 by chromatin immunoprecipitation (ChIP). ChIP was performed with wild-type and Ino80 KO ESC after differentiation as a monolayer for six days in serum containing media lacking 2i + LIF. Position of PCR amplicons are shown in panel a (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). d Cartoon showing the Bmp4 promoter and the location of PCR amplicons used in FAIRE experiments are shown as black bars labeled 1-5. e Changes in chromatin structure ~1.5 Kb upstream of Bmp4 in Ino80 KO ESC. FAIRE was performed with wild-type and Ino80 KO ESC after differentiation in serum containing media lacking 2i + LIF as a monolayer for six days. Position of PCR amplicons is shown in panel d (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). f Increased H3K4me3 occupancy at Bmp4 in Ino80 KO ESC. ChIP was performed with wild-type and Ino80 KO ESC after differentiation as a monolayer for six days in serum containing media lacking 2i + LIF. PCR amplicon 3 from panel b was used for ChIP (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). g Increased SP1 occupancy at Bmp4 in Ino80 KO ESC. ChIP was performed with wild-type and Ino80 KO ESC after differentiation in serum containing media lacking 2i + LIF as a monolayer for six days. PCR amplicon 3 from panel b was used for ChIP (Representative of N = 3 biological replicates; * = t test p ≤ 0.05). h Model for Ino80 function during proximal-distal axis establishment. In wild-type embryos, Ino80 in part functions in the EmE to repress Bmp4 expression. Bmp4 repression in the EmE promotes DVE differentiation. In Ino80 KO embryos, Bmp4 is abnormally expressed in the EmE, which inhibits DVE differentiation. A lack of a DVE prevents the AVE from forming, which subsequently prevents gastrulation. ESC embryonic stem cell, LIF leukemia inhibitory factor, FAIRE formaldehyde assisted isolation of regulatory elements, KO knockout, EmE embryonic ectoderm, DVE distal visceral endoderm, AVE anterior visceral endoderm

Article Snippet: Expanded blastocysts were hatched using Tyrodes solution (Sigma T1788) and allowed to attach and outgrow on gelatinized plates for seven days in defined 2i + LIF media without serum (Millipore Cat# SF016-100).

Techniques: Expressing, Cell Differentiation, Quantitative RT-PCR, Chromatin Immunoprecipitation, Labeling, ChIP-chip, Amplification, Isolation, Knock-Out