title: GSM1149440: WN_3_reads; Yarrowia lipolytica; RNA-Seq
library_construction_protocol: mRNA purified from total RNA using oligo dT Dynabeads (Invitrogen) was fragmented to an average size of 200 nucleotides by a 5 minute heat treatment (70°C) with a buffered zinc solution (Fragmentation Reagent, Ambion). Fragmentation of mRNA was stopped using an EDTA based Stop buffer (Ambion). Fragmented mRNA was incubated with 3 μg Random Hexamer Primers at 65°C for 5 min. First strand cDNA synthesis was carried out using 1 x First Strand Buffer (Invitrogen), 10 mM DTT, 500 μM dNTP mix (Invitrogen), 20 Units RNaseOUT and 200 units SuperScriptTM II Reverse Transcriptase (Invitrogen). For strand-specific library generation, unincorporated dNTPs were subsequently removed using G-50 Micro Columns (GE Healthcare). Second strand cDNA was generated using 1x Second Strand Buffer, 300 μM dNTP mix, 2 units RNaseH, 50 units DNA Polymerase I (NEB), while a 300 μM dUTP mix was used instead of a dNTP mix for the generation of a strand specific library. This material was used for library preparation. For strand-specific libraries, DNA fragments were blunted in an End Repair reaction using T4 DNA Polymerase, Klenow DNA Polymerase and T4 Polynucleotide Kinase, after which a single ‘A’ base was added to the 3’ end using dATP and Klenow Exo Fragment. To allow multiplexing of samples 6-nt barcoded Illumina compatible adapters (AGCTAT, CGATCT, GCTAGT) were ligated to the ends of the DNA fragments, in place of the commercially supplied adapters (PMID:18794863). Fragments of approximately 200-250 base pairs were purified from a 2% Agarose Gel. The second strand containing uridine was removed by treatment with 1 unit Uracil N-glycosylase (UNG) in TE Buffer at 37°C for 15 min. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 13 cycles of PCR. All libraries were quantified using a Qubit® Fluorometer (Invitrogen) and assessed on a 2% agarose gel. Four libraries were generated by GATC and sequenced on an Illumina HiSeq2000