6 protocols
Reads where then mapped to Hg18 by Applied Biosystem's whole transcriptome analysis pipeline (v1.2). Two mismatches were allowed in both seed regions (bases 1-25, and bases 20-45) of the aligned read. Reads that mapped uniquely were retained. Reads which mapped to a filter reference (Applied Biosystems) where removed from further analysis reads. The python package HTSeq (v 0.4.7), was used to count the number of reads mapping to each gene. Expression values (read counts) were obtained by summing the number of reads that mapped uniquely to annotated regions of the Hg18. Reads that mapped to a locus that had more than one annotated gene were considered ambiguous and not used.
Base calling and quality scoring done during SOLiD primary analysis. csfasta and qual files of sequencing reads were retrieved from the sequencing machine. These files were converted to srf format using solid2srf_v0.7.5.
nucleic acid sequencing protocol
Emulsion PCR and sequencing with the SOLiD v.3 system was performed according to the manufacturers protocol (Applied Biosystems, Foster City, CA).
nucleic acid library construction protocol
Total RNA was extracted using the RNAeasy kit (Qiagen, Hilden, Germany). RNA integrity was verified on an Agient 2100 Bioanalyzer. mRNA was isolated with the Dynabeads mRNA DIRECT Kit (Invitrogen, Carlsbad,CA). For each sample, a library for SOLiD sequencing was prepared with the Whole transcriptome analysis kit (4409491 Rev. D, Invitrogen, Carlsbad,CA). Briefly, polyA RNA was fragmented using RNAseIII, purified, hybridized and ligated to RNA adapters. Ligation products were reverse transcribed, purified and ca. 200 nt cDNA fragments selected after electrophoresis on a 6% TBE-Urea gel. cDNA was PCR amplified with SOLiD sequencing primers and purified to produce a library suitable as a template for emulsion PCR. DNA concentrations were determined using qPCR and a pooled library was generated by combining equimolar amounts from all samples.
HCT116 cells were grown in McCoy's 5a medium (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (PAA, Pasching , Austria) without antibiotics
RNAi knockdown in HCT116 cells (ATCC) was performed by reverse transfection in 12 well format. Ambion silencer select RNAi reagents (Ambion) were used at 10nM final concentration using a 1:1000 final dilution ofDharmafect 1 (Dharmaon). Cells wer seeded at 100000 cells/well in a total volum of 1ml and incubated for 3 days. 2 wells were pooled for each treatment.