Supplementary Figure 8: Acute Cas9-dependent deletion of Bcl11b and its cofactors.
From: Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16
(a), Cas9-GFP introduced Scid.adh.2c2 cells were infected with sgRNA-CFP retroviruses. Two days after infection, nuclear lysates of the cells were prepared and subjected to IB with anti-Bcl11b, anti-Chd4, anti-Mta2, anti-Rest, anti-Ring1b, anti-LSD1 and anti-LaminB Abs. (b), Flow cytometry analysis of BM-derived precursors after 7 days of OP9-DL1 culture (before retrovirus infection) is shown. (c), Flow cytometric analysis of sgRNA transduced BM-derived pro-T cells after a total of 14 days of OP9-DL1 culture (7 d after transduction) is shown. Note the high percentage of sgRNA-transduced CFP+ cells. (d), Efficient disruption of Bcl11b cofactors by CRISPR-Cas9 system in primary DN cells. RNA-seq tracks for the cofactor loci are shown. Red arrowheads and dotted lines show sites against which sgRNA was designed. Data are representative of two independent experiments (a, d) or three independent experiments (b, c).
