title: GSM954719: RNA-seq_prmt10; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: 12-day-old seedlings (from MS plates containing 3% sucrose grown under LD at 23°C) expressing either CFP-HLP1 or CFP-HLP1ΔRRM were soaked in ice-cold PBS buffer and irradiated twice for 400 mJ/cm2 in the Hoefer UVC 500 Ultraviolet Crosslinker (GE). CLIP experiment was performed as described (Licatalosi et al., 2008; Xue et al., 2009). Poly(A) site sequencing was performed as described (Fu et al., 2011; Shepard et al., 2011). Briefly, poly(A) RNAs were purified using the mRNA purification kit (Invitrogen), and heat fragmented at 95°C for 30 min. Reverse transcription (Superscript Ⅱ, Invitrogen) was performed using our modified HITS-3’ adaptor at 42°C for 30 min, then add the HITS-5’ adaptor (a SMART oligo) and incubated for additional 30 min. The cDNAs were purified using the Qiagen PCR Cleanup kit and the second strand cDNAs were synthesized by 3 cycles PCR using Phusion DNA polymerase (NEB) and the PE1.0 and PE2.0 primers. The PCR products were separated by 2% agarose gel and the 200-300 bp bands were excised and purified. Gel-extracted DNAs were further amplified by a 13-cycle PCR. The PCR products were purified using the Qiagen PCR Cleanup kit. TA-cloning was performed before Illumina GA Ⅱ sequencing.