8 female patients of age from 27 to 67 years (P1 = 59, P2 = 40, P3 = 27, P4 = 47, P5 = 31, P6 = 35, P7 = 32, P8 = 67) underwent thorough clinical examination including cystoscopy and fulfilled the criteria of European Society for the Study of Interstitial Cystitis (ESSIC) [20]. All patients had an established diagnosis of IC for more than four years. Midstream

urine (30 ml) was collected by the clean catch method with labial separation supervised by an urotherapy nurse. Specimens were kept at 4°C, and within an hour processed for DNA isolation. All specimens used were culture-negative, as tested by the Urological Clinic at the University Hospital HF Aker-Oslo. None of the patients was receiving antibiotics at the time samples were EGFR inhibitor taken, nor prior to that according to hospital MLN2238 price records. Sample processing and DNA isolation Sample processing and DNA extraction was performed as previously described in Siddiqui et al. (2011) [16]. Briefly, urine aliquots (30 ml) were pelleted by centrifugation and total DNA was isolated from sediments using DNeasy

Blood & Tissue kit (QIAGEN, Germany), preceded by incubation with POWERlyse (lysis buffer) (NorDiag ASA, Oslo, Norway). Finally, the DNA was eluted in 100 μl of AE buffer from the kit. The DNA concentrations in the samples (P1-P8) were measured by Quant-iT PicoGreeen dsDNA assay kit (Molecular Probes, Invitrogen USA) and ranged from 0.22 ng/μl to 4.36 ng/μl. 16S rDNA PCR and 454-pyrosequencing For each IC urine sample, we amplified 16S rDNA sequences using two different primer sets specific for the V1V2 and V6 hypervariable regions followed by 454 pyrosequencing as described in Siddiqui et al. (2011) [16]. Each of the primers consisted of a target specific region at their 3’ end (V1V2 or V6) and an adapter sequence (Primer A or Primer B) at their 5’ end as needed for GS FLX amplicon sequencing (454 Life Sciences, USA). Equal amounts of the two different amplicons (both V1V2- and V6-region) for a single subject were pooled and sequenced

using GS-FLX chemistry in the same lane of a Pico-Titer plate PLEK2 divided into 16 lanes, except for samples P1, P2 and, P3, for which each amplicon (V1V2 and V6) was sequenced in a separate lane. 454 pyrosequencing was performed by the Norwegian Sequencing Centre (NSC) at the Department of Biology, University of Oslo, Norway. Sequence read preprocessing Sequence read preprocessing was done as described in Siddiqui et al. (2011) [16]. In brief, a total of 187,901 reads were produced from IC female urine samples. The initial sequence reads were split into two pools using the V1V2 and V6 primer sequences via the mTOR activity sfffile program from 454 Life Sciences (Roche), thus reducing the sequences to 172,931 IC urine reads (Table 1) due to the program splitting on an exact primer match.