GSM3659806: WT_19 [small RNA]; Arabidopsis thaliana; ncRNA-Seq
Following the sorting of pollen grains, whole RNA was isolated using the Direct-Zol™ RNA MiniPrep Kit by Zymo Research (Irvine, California, US) since the specific columns provided in this kit prevent loss of small RNAs. The freshly sorted samples were centrifuged for 3 min at 3,000 rpm and the supernatant removed. Subsequently, the pollen were resuspended in 750 μl Tri-reagent LS (Sigma-Aldrich, St. Louis, US) and flash frozen in liquid nitrogen. The samples were ground in a cooled mortar to break up pollen grains until the samples liquefied and transferred to fresh reaction tubes. Afterwards, the protocol was followed according to the manufacturer's specification. RNA concentration was measured using a NanoDrop and analysed on a BioAnalyzer to verify the existence of small RNAs as well as to check for possible RNA degradation. Generation of small RNA libraries from three paired pollen RNA isolations (wild-type and paps1-3 mutant pollen sorted from the same sample) and sequencing was carried out by Eurofins Medigenomix GmbH, Ebersburg, Germany. Complementary DNA (cDNA) libraries were constructed containing fragments with a length of approximately 19-25 bp with TruSeqTM Small RNA Sample Preparation Kit (Illumina, San Diego, US) and sequencing was performed using Illumina HiSeq 2500.
GEO Accession
GSM3659806