Project description:Purpose: Analyses of transcriptomes of WT and pumpkin mutants, in order to compare gene expression of typically NEP or PEP-transcribed plastid genes.
Project description:Stroma extracts were isolated from 2-week-old WT plants and incubated with either PUMPKIN-specific antibodies or with the pre-immune serum. IgGs were captured with SiMAG-Protein G beads (Chemicell) and recovered RNA was used for generation of libraries with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench, the mean RPKM values of the replicates as well as the ratio of PUMPKIN vs Pre-immuneserum were calculated. Five prominent RNA targets (trnG-UCC, trnV-UAC, petB, petD and ndhA) were identified and validated via Slot Blot analyses.
Project description:To demonstrate RIPSeeker program that is developed for RIP-seq analyses, we generated RIP-seq data corresponding to the protein CCNT1 in HEK293 cell line using standard RIP-seq protocols described in Zhao et al., (2010). We performed two in-house RIP-seq experiments both for CCNT1 in human HEK293 cells. Briefly, we generated tagged CCNT1 using a triple tag system that supports lentiviral stable expression and mammalian affinity purification (MAPLE) Mak et al (2010). The HEK293 cells stably expressing tagged CCNT1 was purified by M2 agarose beads, followed by RNA extraction by Trizol. The library synthesis was carried out according to the RIP-seq protocol described in Zhao et al., (2010) except that one of the two experiments was done with non-strand-specific sequencing.