GSM1074244: PAS-seq_hlp1_prmt10_double_mutant_rep2; Arabidopsis thaliana; OTHER
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.
GEO Accession
GSM1074244