GSM2087992: PCF RiboProf 427 replicate 1; Trypanosoma brucei; RNA-Seq
For in vivo blood form parasites, the blood from 2-3 infects rats was pooled and centrifuged and the buffy coat extracted and placed into 20 ml HMI-9 without serum pre-warmed to 37°C.  To arrest translation, cycloheximide was added to 100 μg/ml and incubated for 2 minutes at 37°.  To rapidly chill the cells, 300 ml of ice-cold phosphate buffered saline with glucose (PSG) was added and the cells were pelleted at 4°.  Only parasite populations with greater than 99% slender cells were used.  In vitro grown parasites were pelleted at 5000g and resuspended in 50 mls of media lacking serum.  Cycloheximide was added to 100 mg/ml at the cell incubated for 2min at room temperature (procyclic cells) or 37° (bloodform cells). Following cycloheximide treatment the cells were rapidly chilled with PSG and pelleted at 5000g.  Lysates from both type of parasite pellets were then prepared as following. Cell pellets were resuspended in Buffer A (10 mM Tris pH 7.4, 300 mM KCl, 10 mM MgCl2 plus protease inhibitors to approximately 1.3x109/ml.  Approximately one-third of the sample was placed into TRIzol (Life Technologies) for RNA extraction following manufacturer’s suggested protocol.  To the remainder, one-sixth volume of buffer A containing 0.2M sucrose and 1.2% Triton N-101 was added and the samples were dounced (30 strokes in a chilled homogenizer). After transfer to a prechilled microfuge tube, the samples were clarified by centrifugation in a microfuge at 15000 rpm for one minute.  The supernatant was withdrawn, pooled if needed, and then aliquots flash frozen in liquid nitrogen for storage at -70°C. After thawing on ice, RNase I (Ambion) was added at 30 units/OD260 of lysate.  Samples were then incubated for 1 hour at room temperature.  RNase digestion was stopped by adding 400 units RNasin (Promega).  Samples were them layered over a 1 ml 1M sucrose cushion prepared in buffer A and ribosomes were pelleted by centrifuging for 4 hours at 70,000g in an SW55 rotor.  After removing the supernatant, the ribosomal pellet was resuspended in 500 μl buffer A with the MgCl2 replaced with 10 mM EDTA to dissociate the ribosomes and deplete larger rRNA fragments.  The protected fragments were then separated from contaminating ribosomal RNA by passage through an Amicon Ultra-4 or YM-100 column with 100,000 MW cutoff. After more than 400 μl had flowed through the membrane the RNA in the flow-through was extracted with phenol:CHCl3:iAA and the RNA precipitated.  Poly(A)+ RNA was isolated using Dynabeads mRNA Direct (Life Technologies).  RNA was fragmented as described Ingolia et all 2011.  Ribosomal protected fragments between 28 and 31 nucleotides and fragmented Poly(A)+ RNA between 30 and 70 nucleotides was isolated from 15% accrylimide gels and libraries prepared for sequencing.  For detailed protocol on generating sequencing libraries for both the ribosome protected and fragmented mRNA library see Ingolia et a 2011.  Briefly, following dephosphorylation the adapter Linker-1 (IDT) was ligated to the 3’ end of the fragment and the ligated product gel purified.  The adapter was used for priming reverse.  Following gel purification the cDNA was circularized with Circ Ligase (Epicenter Biotechnologies).  Circles containing ribosomal RNA were subtracted using biotinylated primers .  The final library was generated by PCR.To map the 5’ end of the transcripts, libraries enriched for the 5’ ends of mRNAs were constructed from three biological samples of strain 927 (2 PCF and 1 slBF selected from the biological samples above).  SL libraries were constructed as described Mittra B et al 2013.  In brief, RNA was prepared and cDNA synthesized using primer ending in a random 6 bases.  Second strand synthesis was primed using a primer that matches the T. brucei SL sequence.  The sequencing library was generated by PCR. Duplexed libraries were sequenced using Illumina GA II machines at the High Throughput Genomics Unit at the University of Washington to generate ~36 nt reads.
GEO Accession
GSM2087992