title: GSM2151885: RNA from GFP+ protoplasts replicate 3; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: To generate material for fluorescence-activated cell sorting, we crossed the ap1 cal mutant (Nottingham Arabidopsis Stock Centre accession N6161) to the DRNL::erGFP marker line and identified transgenic DRNL::erGFP ap1 cal progeny in the F2 generation. The TruSeq v2 RNA sample preparation kit (Ilumina) was used to prepare cDNA libraries starting from 200 ng total GFP+ or GFP- RNA. Poly(A)+ RNA was purified onto oligo-dT magnetic beads and was fragmented using divalent cations at elevated temperature; RNA fragments were reverse-transcribed using random primers, followed by second-strand cDNA synthesis with RNase H/DNA Polymerase I. After end repair and A-tailing, adapters were ligated and the indexed cDNA products were purified and amplified by PCR (15 cycles) to create the final cDNA libraries. Library quality was validated on a 2200 TapeStation (Agilent Technologies) and individual libraries were quantified on the Qubit System (Invitrogen) prior to pooling and pool quantification via the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems). The pooled, indexed libraries were loaded and analysed on a HiSeq sequencer (Illumina) using the 2 × 100-bp v3 protocol.
attributes.tag: GEO Accession
attributes.value: GSM2151885