title: GSM2695708: RNA ΔH3.V/ΔH4.V R1; Trypanosoma brucei brucei; RNA-Seq
library_construction_protocol: Total RNA from 45 million cells was extracted in triplicates (NucleoSpin RNA, Macherey & Nagel) and spiked with a synthetic RNA control (ERCC, invitrogen). rRNA was depleted from the sample using custom-made anti-rRNA oligos that were hybridized to the targed RNA and subsequently digested with RNAseH. Residual DNA was digested away with a DNAse and the sample was purified using RNeasy Minelute columns (Qiagen). Libraries were prepared as follows: cDNA was produced from 100 ng of rRNA depleted RNA using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® until second strand cDNA synthesis. The double-stranded cDNA was purified using Agencourt AMPure XP Beads and eluted in 0.1X TE Buffer. cDNA was end-repaired using T4 DNA polymerase (NEB), Klenow large Fragment (NEB) and  T4 DNA Polynucleotide Kinase (NEB). Protruding A-bases were added to the 3` end using  Klenow fragment (3´ to 5´ exo minus) and dATP. TruSeq adapters were ligated to the cDNA fragments using Quick Ligase (NEB) and was purified using AMPure XP magnetic beads. Y-shaped adapters were converted to dsDNA with Illumina primers for 5 cycles, purified with magnetic beads  and library fragments of 200-500 bp were size selected from an agarose gel. The gel-purified library was further amplified for 10-17 cycles using Illumina primers and purified using magnetic beads. Libaries were quantified using Qubit and qPCR and were sequenced on an Illumina flow cell  in an Illumina NextSeq aparatus following the manufacturer's instructions
attributes.tag: GEO Accession
attributes.value: GSM2695708