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Supplementary figure. Promoter use for Xist transcription in wild type and Xist^1lox testis. (a) Despite deletion of P₁, a strand-specific Xist RTPCR product is still seen using primers JT4/MIX20 in Xist^1lox testis, but is absent from Xist^trun testis which lacks exon 4 in which the JT4 primer is located. The Xist^1lox transcript is regulated in the same way as wild type Xist (see Fig. 2a), being barely detectable in male heart and undetectable in other tissues such as liver. (b) Transcripts originate upstream of P₁ in wild type and Xist^trun testis, as shown by strand-specific Xist RTPCR using primers CJ3 (5¢ CAT ACC TTG ATG TAC ACG GT 3¢) and CJ4 (5¢ TGG ATA TGG CTT TGT ATA AA) that span from 1247 to 1470 bp upstream of P₁ (product=223 bp). Xist^1lox testis provides a negative control because the CJ3/CJ4 primers lie within the region deleted in this mutant. No transcript can be detected at this site in adult male liver or in ES cells. Methods: Strand-specific PCR was as described in Materials and Methods, except that for primers CJ3 and CJ4 samples were divided into +RT and –RT reactions and processed in parallel. All --–RT samples showed no amplification.

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