Bovine serum albumin (bsa)

For the isolation of tumor-bearing lungs from KP mice, mice were anesthetized, perfused with PBS as described, and 4% paraformaldehyde (PFA; Fisher Scientific) was added to the lungs via intratracheal instillation. Lungs were then removed, placed in 4% PFA overnight at 4°C, and stored in 70% ethanol prior to processing. The fixed lung tissue was then paraffin embedded at the UCSD Histology and Immunohistochemistry Core at The Sanford Consortium for Regenerative Medicine according to standard protocols, and 5 μm sections were obtained. Paraffin embedded tissues were then deparaffinized in xylene and rehydrated in a 100%, 95%, 70%, 50%, and 30% ethanol series. For immunohistochemistry, endogenous peroxidase was quenched in 3% hydrogen peroxide (Fisher Scientific) for 30 min at room temperature prior to antigen retrieval. Antigen retrieval was performed for 30 min in 95–100 °C 1× citrate buffer, pH 6.0 (eBioscience). Sections were blocked in TBS (Tris buffered saline) or PBS containing 0.1% Triton X-100 (Sigma-Aldrich), 10% goat or donkey serum (Sigma-Aldrich), and 5% bovine serum albumin. For single-cell suspensions of lung epithelia or lung tumors, Lin⁻EpCAM⁺ cells were isolated via FACS in the manner described previously. Cells were resuspended in DMEM:F12 (Fisher Scientific) supplemented with 50% FBS and adhered to slides by centrifugation at 500 rpm. Twenty-four hours later, cells were fixed in 4% PFA (Fisher Scientific), washed in PBS, and blocked with PBS containing 0.1% Triton X-100 (Sigma-Aldrich), 10% Goat serum (Fisher Scientific), and 5% bovine serum albumin (Invitrogen).
All incubations with primary antibodies were carried out overnight at 4°C. For immunofluorescence staining, incubation with Alexafluor-conjugated secondary antibodies (Molecular Probes) was performed for 1 hr at room temperature. DAPI (Molecular Probes) was used to detect DNA and images were obtained with a Confocal Leica TCS SP5 II (Leica Microsystems). For immunohistochemical staining, incubation with biotinylated secondary antibodies (Vector Laboratories) was performed for 45 min at 20–25°C. Vectastain ABC HRP Kit (Vector Laboratories) was used according to the manufacturer’s protocol. Sections were counterstained with hematoxylin. The following primary antibodies were used: chicken anti-GFP (Abcam, ab13970; 1:200), rabbit anti-Msi2 (Abcam, ab76148; clone EP1305Y, 1:500), rabbit anti-SPC (Santa Cruz, sc-13979; clone FL-197, 1:100), and goat anti-CC10 (Santa Cruz, sc-9773; clone S-20, 1:200). Whole slide imaging was used to determine the number of tumors, tumor burden, and tumor grade in the tumor-bearing lungs of KP mice. H&E-stained slides were scanned using Aperio AT2 digital whole slide scanning (Leica Biosystems), and tumor analysis was performed using ImageScope (Aperio). Tumor grade was scored according to the criteria described by Jackson et al., 2005 (link).

Extracted DNA from the 0.2–3 µm size-fraction was amplified in three independent 50 µL aliquots per domain using full-length 454 primers. PCR reactions contained: 1X HF buffer (NEB), 200 µM of each dNTP (Feldan Bio), 0.4 mg/mL BSA (Fermentas), 0.2 µM of each 454 primer (Invitrogen), 1 U of Phusion High-Fidelity DNA polymerase (NEB), and 1–3 µL of template DNA. Three separate DNA concentrations were used for each sample: 1/0.5/0.1X for Bacteria and Eukarya; and between 3X and 0.1X for Archaea that represent the smallest proportion of microbial biomass in the Beaufort [18] . Cycling conditions were as follows: an initial denaturation at 98°C for 30 s, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. The triplicate reactions for the separate domains were pooled, purified using the QIAquick PCR purification kit (QIAGEN), and quantified spectrophotometrically (Nanodrop ND-1000). The 11 sample-coded amplicons were mixed in equal quantity and 1/8^th plate for each domain was sequenced on a Roche 454 GS-FLX Titanium platform at the McGill University/Génome-Québec Innovation Centre for Bacteria and at the IBIS/Université Laval Plate-forme d'Analyses Génomiques for Archaea and Eukaryotes. The raw pyrosequencing reads have been deposited in the NCBI Sequence Read Archive with accession number SRA029114 and a “Minimal Information about a MARKer gene Sequence” (MIMARKS) compliant table is included with the data and given here in Table S3.

HCT116 or K562 cells (1×10⁶) were transfected in triplicate with Bi-miR-34a or Bi-cel-miR-67 (Dharmacon) as described above and then cultured in six-well plates. Twenty-four hours later, the cells from 3 wells were pelleted at 500×g. After washing twice with PBS, cell pellets were resuspended in 0.7 ml lysis buffer (20 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 0.3% NP-40, 50 U of RNase OUT (Invitrogen), complete mini-protease inhibitor cocktail (Roche Applied Science)), and incubated on ice for 5 min. The cytoplasmic lysate was isolated by centrifugation at 10,000×g for 10 min. Streptavidin-coated magnetic beads (Invitrogen) were blocked for 2 hr at 4°C in lysis buffer containing 1 mg/ml yeast tRNA and 1 mg/ml BSA (Ambion) and washed twice with 1 ml lysis buffer. Cytoplasmic lysate was added to the beads and incubated for 4 h at 4°C before the beads were washed five times with 1 ml lysis buffer. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated using Trizol LS reagent (Invitrogen). The level of mRNA in the Bi-miR-34a or Bi-cel-miR-67 control pull-down was quantified by qRT-PCR or mRNA microarray. For qRT-PCR, mRNA levels were normalized to a housekeeping gene (GAPDH, SDHA or UBC). The enrichment ratio of the control-normalized pull-down RNA to the control-normalized input levels was then calculated.

Except for the ligation step, Hi-C was performed essentially as described in Lieberman-Aiden et al. [15 (link)], with some modifications.
Thirty to 50 million cells were fixed in 2 % formaldehyde for 10 min, quenched with 0.125 M glycine, spun down (400 × g, 5 min) and washed once with phosphate-buffered saline. The cells were incubated in 50 ml permeabilization buffer (10 mM Tris–HCl pH 8, 10 mM NaCl, 0.2 % Igepal CA-630, Complete EDTA-free protease inhibitor cocktail [Roche]) for 30 min on ice with occasional agitation, spun down (650 × g, 5 min, 4 °C), and the cell pellets were resuspended in 358 μl of 1.25× NEBuffer2 (NEB) per 5 million cell aliquot. We added 11 μl of 10 % SDS to each aliquot, followed by an incubation at 37 °C for 60 min with continuous agitation (950 rpm). To quench the SDS, 75 μl of 10 % Triton X-100 was then added per aliquot, followed by an incubation at 37 °C for 60 min with continuous agitation (950 rpm). To digest chromatin, 1500 U of HindIII (NEB) was added per aliquot and incubated at 37 °C overnight with continuous agitation (950 rpm). After digestion, restriction sites were filled in with Klenow (NEB) in the presence of biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (all 30 μM) for 60 min at 37 °C.
For in-solution ligation, 86 μl of 10 % SDS was added per aliquot and incubated at 65 °C for 30 min with continuous agitation (950 rpm), followed by addition of 7.61 ml of ligation mix (745 μl of 10 % Triton X-100, 820 μl of 10× T4 DNA ligase reaction buffer [NEB], 82 μl of 10 mg/ml bovine serum albumin [NEB] and 5.965 ml water) per aliquot and incubation at 37 °C for 60 min with occasional agitation. For in-nucleus ligation, 7.61 ml of ligation mix (820 μl of 10× T4 DNA ligase reaction buffer [NEB], 82 μl of 10 mg/ml bovine serum albumin [NEB] and 6.71 ml water) was added per aliquot (compared with the in-solution ligation, SDS addition and incubation at 65 °C were omitted). For the ligation reaction (both in-solution and in-nucleus variants), 50 μl of 1 U/μl T4 DNA ligase (Life Technologies) was added per aliquot, followed by incubation at 16 °C for 4 h.
The cross-links were reversed by adding 60 μl of 10 mg/ml proteinase K (Roche) per aliquot and incubating at 65 °C overnight. After overnight incubation, another 60 μl of proteinase K per aliquot was added, followed by incubation at 65 °C for an additional 2 h. RNA was removed by adding 12.5 μl of 10 mg/ml RNase A (Roche) per aliquot and incubating at 37 °C for 60 min. DNA was isolated by a phenol (Sigma) extraction, followed by a phenol/chloroform/isoamylalcohol (Sigma) extraction and standard ethanol precipitation. The precipitated DNA was washed three times with 70 % ethanol, and dissolved in 25 μl TE per aliquot. Subsequently, all aliquots were pooled and the Hi-C DNA was quantified (Quant-iT Pico Green, Life Technologies). Biotin was removed from non-ligated restriction fragment ends by incubating 30–40 μg of Hi-C library DNA with T4 DNA polymerase (NEB) for 4 h at 20 °C in the presence of dATP. After DNA purification (QIAquick PCR purification kit, Qiagen) and sonication (Covaris E220), the sonicated DNA was end-repaired with T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB) and dNTPs in 1× T4 DNA ligase reaction buffer (NEB). Double size selection of DNA was performed using AMPure XP beads (Beckman Coulter), before dATP-addition with Klenow exo⁻ (NEB). Biotin-marked ligation products were isolated with MyOne Streptavidin C1 Dynabeads (Life Technologies) in binding buffer (5 mM Tris pH8, 0.5 mM EDTA, 1 M NaCl) for 30 min at room temperature, followed by two washes in binding buffer, and one wash in 1× T4 DNA ligase reaction buffer (NEB). Paired-end (PE) adapters (Illumina) were ligated onto Hi-C ligation products bound to streptavidin beads for 2 h at room temperature (T4 DNA ligase in 1× T4 DNA ligase reaction buffer [NEB], slowly rotating). After washes in wash buffer (5 mM Tris, 0.5 mM EDTA, 1 M NaCl, 0.05 % Tween-20) and binding buffer, the DNA-bound beads were resuspended in NEBuffer 2. Bead-bound Hi-C DNA was amplified with 12 PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). The concentration and size distribution of Hi-C library DNA after PCR amplification was determined by Bioanalyzer profiles (Agilent Technologies) and quantitative PCR, and the Hi-C libraries were paired-end sequenced on Illumina Hi-Seq 1000 or MiSeq platforms.