title: GSM1074247: PAS-seq_VLD_to_AAA_mutant_rep1; Arabidopsis thaliana; OTHER
library_construction_protocol: The seedlings were ground into fine power in liquid nitrogen and RNA was extracted by TRNzol reagent (Tiangen). Poly(A) site sequencing was performed as described with modifications at the HITS-3’ adaptor and sequencing primer (Fu et al., 2011; Shepard et al., 2011). Briefly, poly(A) RNAs were purified using a mRNA purification kit (Invitrogen), and heat fragmented at 95°C for 30 min. Reverse transcription (Superscript, Invitrogen) was carried out using our modified HITS-3’ adaptor at 42°C for 30 min, then the HITS-5’ adaptor (a SMART oligo) was added and incubated for an additional 30 min. The cDNAs were purified using a Qiagen PCR Cleanup kit and second strand cDNAs were synthesized by 3 cycles of PCR using Phusion DNA polymerase (NEB) and the PE1.0 and PE2.0 primers. The PCR products were separated on a 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 a Qiagen PCR Cleanup kit.