The osteosarcoma cell line U-2 OS (ATCC-LGC Promochem, Borås, Sweden) was cultivated in a 5% CO2 environment at 37°C in McCoy's 5A media, as suggested by the provider, with the addition of 10% Fetal Bovine Serum (FBS) and an antibiotic/antimycotic solution (both from Invitrogen).
RNA sample preparation and cDNA synthesis
Cells were harvested and RNA was extracted using the RNeasy extraction kit as instructed by the manufacturer (Qiagen) and quality-assessed using the RNA nano kit on a BioAnalyzer 2100 (Agilent). The BioAnalyzer can interpret the generated data and score it with a RNA Integrity Number (RIN) ranging from 1 (very degraded) to 10 (no degradation). 10 μg of high-quality (RIN >9.5) total RNA was used as input material for depletion of ribosomal fragments using RiboMinus (Invitrogen). 250 ng ribosome-depleted RNA was adjusted to 4.5 μl in nuclease-free water and fragmented in 95°C for 20 minutes, after which it was immediately transferred to ice. 1 μl of biotinylated tagged random hexamers (Biotin-TEG-CTTTCCTCTCTATGGGCAGTCGGTGATNNNNNN, 1 pmol/μl, Operon) was added and the mixture was denatured at 70°C for 10 minutes and on ice for 2 minutes. During incubation, a cDNA-synthesis master mix consisting of (per sample) 6 μl 5x First-strand buffer (Invitrogen), 3 μl 0.1 M DTT (Invitrogen), 7.5 μl dNTP mixture (2 mM/dNTP) and 6.5 μl nuclease-free water was assembled on ice. Of this master mix, 23 μl was added to the denatured RNA:hexamer mixture together with 2 μl SuperScript III (Invitrogen) on ice. The first strand cDNA synthesis reaction was incubated at 20°C for 10 minutes followed by 37°C for 10 minutes and 42°C for 45 minutes. First-strand cDNA was purified by addition of 70 μl nuclease-free water using a MinElute spin column following the manufacture's instructions (Qiagen). Elution was carried out twice, each in 10 μl of EB-buffer, with a 180° rotation of the column in the centrifuge between the elutions. A second strand synthesis master mix was assembled on ice. This consisted of (per sample) 79 μl nuclease-free water, 30 μl 5x second-strand buffer (Invitrogen), 15 μl dNTP mixture (2 mM/dNTP), 1 μl 10 U/μl E. coli DNA Ligase (Invitrogen), 4 μl 10 U/μl E. coli DNA polymerase I (Invitrogen) and 1 μl 2 U/μl RNase H (Invitrogen). Of this, 130 μl was added to the ≈18 μl eluate containing the RNA:cDNA hybrid. The reaction was incubated at 16°C for 2 hours, after which 1.5 μl 3 U/μl T4 DNA Polymerase (New England Biolabs) was added, and the reaction was incubated for 5 more minutes at 16°C.
Enrichment and SOLiD DNA sequencing
20 μl of Streptavidin Dynabeads M-270 (Invitrogen) were washed in 50 μl 1× BW-buffer (20 mM Tris, 2 mM EDTA, 1 M NaCl) and pelletized using a MPC-6 magnetic particle concentrator (Invitrogen). To the pellet, the 150-μl second-strand syntheis reaction and 150 2× BW buffer were added, mixed by gentle vortexing and incubated on gentle rotation using a RotaMix (Elmi) at room temperature (≈22°C) for 15 minutes. The bead-DNA complex was then washed three times in 100 μl sterile deionised water and pelletized. A PNK-master mix consisting of (per reaction) 15 μl sterile deionised water, 2 μl PNK buffer (Invitrogen), 2 μl 10 mM ATP and 1 μl 10 U/μl PNK was assembled on ice and 20 μl was added to the pelletized beads and mixed by pipetting. The reaction was incubated at room temperature for 15 minutes. To create a blunt-ended dsDNA adapter sequence suitable for ligation to the beads, a mixture consisting of 440 μl sterile deionised water, 5 μl 100 pmol/μl RDV primer (AACTGCCCCGGGTTCCTCATTCTCT, MWG-Biotech), 5 μl 100 pmol/μl aRDV primer (AGAGAATGAGGAACCCGGGGCAGTT, MWG-Biotech) and 50 μl PNK buffer (Invitrogen) was assembled and incubated at 95°C for 3 minutes and allowed to cool to room temperature on a lab bench for 30 minutes. A ligation master mix consisting of 4 μl 5× Ligase buffer (Invitrogen), 1 μl 1 pmol/μl RDV:aRDV duplex, 14 μl sterile deionised water and 1 μl 3 U/μl T4 DNA Ligase was assembled on ice, added to the pelletized beads and mixed by pipetting. The reaction was incubated for 16 hours on a RotaMix at room temperature. The ligation reaction was washed three times in sterile deionised water and the beads were resuspended in 20 μl of sterile deionised water.
An amplification master mix was assembled, consisting of 5 μl 5× HF buffer (Finnzymes), 10 μl sterile deionised water, 5 μl dNTP mix (2 mM/dNTP), 1 μl 10 pmol/μl RDV primer (AACTGCCCCGGGTTCCTCATTCTCT, MWG-Biotech), 1 μl 10 pmol/μl LAmpFDV (CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT, MWG-Biotech) and 1 μl 2 U/μl Phusion polymerase (Finnzymes). 23 μl amplification master mix and 2 μl beads were mixed denatured at 95°C for 30 seconds and cycled as follows: 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C for 16 cycles. After a final extension at 72°C for 10 minutes, the PCR product was purified using a MinElute column, following the manufacturers instructions with the elution step as described earlier.
The PCR product was subjected to emPCR and SOLiD sequencing following the manufacturers instructions (Life Technologies/Applied Biosystems). Fifteen million 35-base pair reads passed quality filters including filtering against adaptors.
Mapping of DNA reads and defining the transcriptome
The reads were mapped to the human genome (hg18) following the manufacturers instructions. A read was considered to be unique if is mapped to a single location with N mismatches and nowhere with N+1 or N+2 mismatches (i.e. a clear zone of 2). In total, 6 442 847 reads aligned uniquely to EnsEMBL genes. Only unique reads with a maximum of three mismatches were used to calculate expression values. A gene was considered present if at least one read fell entirely inside an exon of the gene. Raw sequence data has been deposited to the NCBI Short Read Archive with accession number SRA023713.1.
Production of Antibodies
The antibodies used in this study have been generated within the Human Protein Atlas program http://www.proteinatlas.org. For each application using the generated antibodies, a standard set of categories has been established, and these are grouped into three main validation scores: (i) supportive, (ii) uncertain, or (iii) non-supportive [Additional file 1: Supplemental table S1] .
Western blot analysis
Prior to immunohistochemistry and immunofluorescence the HPA antibodies were analysed by Western blot as previously described . All membranes were incubated with the primary antibodies diluted 1:500. The secondary HRP-conjugated antibody (Swine Anti-Rabbit Immunoglobulin/HRP, DakoCytomation) was diluted 1:3000 and detection was carried out using a CCD-camera. Seven categories (1-87) for the Western blot validation have been proposed in which three (grade 1-3) are supportive, two (grade 4-5) are uncertain, and two (grade 6-7) are non-supportive.
Immunohistochemistry was performed on cell microarrays (CMA) where U-2 OS cells were represented, as previously described . In brief, cells were harvested, fixed in formalin and dispersed into agarose. After histoprocessing and paraffin embedding of the cell pellets resulting in donor blocks, duplicate 0.6 mm punches were sampled and put into one recipient CMA. 4 micron sections were subsequently cut and immunohistochemically stained using an Autostainer Plus instrument (Dako, Glostrup, Denmark). An automated slide-scanner system, Scanscope T2 (Aperio Technology, Vista, CA, USA) was used to image the stained sections, resulting in digital images representing separated cell spots from the CMA.
The images were analyzed by an automated image analysis software, TMAx (Beecher instruments, Sun Praire, WI, USA), which identifies cells, measures the immunostaining and counts the fraction of stained cells, all according to processing logic previously described . The measured staining intensity was further categorized into a IHC graded scale (negative, weak, moderate and strong). Here, the analyzed protein was considered "absent" when the staining intensity score was negative and "present" when the staining intensity score was weak, moderate or strong.
Immunofluorescent stainings were performed as previously described . Briefly, cells were seeded in 96-well glass bottom plates, fixed with paraformaldehyde and permeabilized with Triton X-100 before immunofluorescently stained. The entire procedure was automated using a pipetting robot. Besides the HPA antibody staining, organelle markers for microtubules, endoplasmic reticulum and nuclei were included. Image acquisition was performed manually using a LSM 510 Meta confocal laser-scanning microscope equipped with a 63x oil-immersion objective (Carl Zeiss GmbH, Jena, Germany). For each sample, two representative four-channel images were acquired. The laser power and detector gain were adjusted for each sample to obtain as good signal to noise ratios as possible and to use the entire dynamic range of the detector.
The images were visually inspected and annotated in terms of staining intensity and subcellular localization. A graded scale (negative, weak, moderate and strong) was used to categorize the staining intensity based on the used laser power and detector gain. For each antibody a validation score was set based on how well the observed subcellular localization agreed with information in the UniProt database. The IF validation scores consist of a nine-graded scale that can be merged into three main categories: supportive, uncertain or not supportive [Additional file 1: Supplemental table S1]. Here, the analyzed protein was considered "absent" when the validation score was 7 (no staining, see [Additional file 1: Supplemental table S1]) and "present" otherwise.