4sU-seq analysis for tethering hnRNPK to Xist lacking of XR-PID

Xist lacking of XR-PID (B-repeat and part of C-repeat) can''t recruit the polycomb during X chromsome inactivation. The majoring of Xist-mediated chromosomal silencing wouldn't be achieved. We found that hnRNPK was the one of main contributors for binding B-repeat of Xist. When we tethered hnRNPK to Xist which lacks of XR-PID region, the silencing and polycomb recruitment are restored. Overall design: Cell lines were generated by randomly integrating Xist lakcing of XR-PID (mainly B-repeat) but with bgl sequence. The fused hnRNPK with bglG were engineered and consitutely expressed. After 3 days of Dox induced Xist RNA expression, 4sU were incorperated into the medium and incubated for 12 minutes, and 4sU-RNAs were isolated and then library preped for sequencing.