GSM4333061: RPF.24.3; Arabidopsis thaliana; OTHER
Pollen was scrapped from the plates and grinded in liquid nitrogen before homogenization in an optimized ice-cold extraction buffer containing 0.1% Tris-HCl pH8, 1% sodium deoxycholate, 40 mM KCl, 20 mM MgCl2, 10% polyoxyethylene-10-tridecyl ether, 1mM DTT, 100 µg/mL cycloheximide, 10U/mL DNAse I. After extraction and centrifugation, the supernatants were transferred into a pre-chilled tube and separated in 100 µL (for RNA library) and 200 µL (for RPF library) according to the Illumina® TruSeq® Ribo Profile Mammalian kit. The ribosomes for RPF libraries were purified, after nuclease digestion, by size exclusion columns using MicroSpin S-400 columns. For rRNA removal, the Ribo-Zero Magnetic bead kit was used. Heat fragmentation of total RNA, end repair of both RNA and RPF as well as all downstream steps: 3' adapter ligation, adaptor removal, reverse transcription, PAGE purification of cDNA, and cDNA circularization were done following strictly the Illumina kit protocol. cDNA Libraries were amplified by 11 or 13 PCR cycles for RNA and RPF respectively, and after gel purification and quantification, equimolar (5nM) pools of libraries were prepared for NGS. Riboprofiling according to the the Illumina® TruSeq® Ribo Profile Mammalian kit
GEO
GSM4333061
GEO Accession
GSM4333061