Extended Data Fig. 4: Allele-specific RNA tagging and ablation establish essential nature of the antisense RNA without interfering with POL-II transcription.
From: Decapping enzyme 1A breaks X-chromosome symmetry by controlling Tsix elongation and RNA turnover
a, Schematic of 3xPP7 (mus allele) and 3xMS2 (cas allele) insertions into Tsix. CRISPR/Cas9 targeted mus-Tsix first. PCR primers for clone screening is indicated (Primers 1 and 2), along with XcmI restriction site. b, PCR-screening showing three positive heterozygous clones for 3xPP7 mus-Tsix insertion. Representative result from 3 independent experiments shown. c, PCR-screening for cas-Tsix insertion of 3xMS2 in C11 clone identified in (b). Positive clones E20 (Clone B) and P20 (Clone A) shown. Representative result from 3 independent experiments shown. d, XcmI digestion confirms the insertion of 3xPP7 (390 bp, mus) and 3xMS2 (digestion pattern: 120+252 bp, cas) into clones E20 and P20. Representative result from 3 independent experiments shown. (e) Allele-specific Tsix RNA levels 6 hours post-LNA nucleofection for independent Clone B. Description as in Fig. 3b. f, Allele-specific Xist RNA level analysis upon Tsix knockdown shown in panel (a) and 24h post nucleofection. Normalized to scramble control. g, Allele-specific Tsix RNA levels 1 and 3 hours post-LNA nucleofection (Clone B). One LNA targeting Tsix mus (PP7) and two LNAs targeting cas (MS2) were used. Normalized to scramble control. h, Allele-specific Xist RNA levels upon Tsix knockdown show a rapid and stable Xist induction at 1 and 3 hours post-nucleofection of Clone B. Xist upregulation is maintained 48-72 hours post-nucleofection. Normalized to scramble control. i, Quantification of Xist RNA clouds upon Tsix knockdown for the analysis in Fig. 3d. Number of nuclei (n) scored across two independent experiments for each clone as shown. In total, 4 biological replicates have been done for this assay with two independent experiments performed for each clone. j, Allele-specific ChIP-qPCR for POL-II-S2- and -S5 phosphorylated isoforms at indicated positions. Negative control IgG pulldown also shown. PCR using primers upstream of LNA-targeted region (Tsix E3). An intergenic region served as negative control (Xneg). Mean and s.e. are indicated, n=3 independent replicates.
