Phenol chloroform

The DNA solution was mixed with an equal volume of phenol-chloroform (Invitrogen, Calif., USA) and vortexed for 1min. The mixture was centrifuged at 15,000 × g for 10min to separate the aqueous and solvent phases. The desired aqueous layer was carefully removed and mixed with an equal volume of phenol-chloroform (1:1) (Invitrogen, Calif., USA) and vortexed for 1min. The mixture was again centrifuged at 15,000 × g for 10min. The aqueous layer was removed and 1/10 volume of chilled 3 M sodium acetate (pH 5.2) and 1/10 volume of isopropanol were added to the DNA sample and mixed well by inverting the tube multiple times. Then, the sample was incubated at −20 °C for 2 h to precipitate the DNA. The DNA was pelleted by centrifugation at 15,000 × g for 30min at 4 °C. The supernatant was carefully removed and 1 ml of cold 70% ethanol was added to wash off the remaining salts. The DNA pellet was allowed to air dry for 5min with the lid opened. The desired amount of TE buffer was added into the tube in order to dissolve the DNA

DNA from fecal and cecal samples were isolated using a bead-beating phenol: chloroform extraction method followed by DNeasy Blood & Tissue Kit (QIAGEN, USA). In short, samples were weighed between 10-50mg and combined with acid-washed glass beads (212-300mm; Sigma-Aldrich, USA), 500uL Buffer A (200 mM NaCl, 200 mM Tris, 20 mM EDTA, 210 uL SDS (20% w/v, filter-sterelized), and 500 uL phenol:chloroform (Thermo Fisher Scientific, USA). The samples were disrupted using a Mini-Beadbeater-16 (Biospec Products, USA) for 3 minutes at room temperature and centrifuged (10,000 rpm, 4°C, 3 minutes). The aqueous phase was recovered and mixed with an equal volume of phenol:chloroform by gentle inversion and centrifuged (10,000 rpm, 4°C, 3 minutes). The remaining aqueous phase was recovered and mixed with 500 μL of chloroform, mixed by gentle inversion, and centrifuged (10,000 rpm, 4°C, 3 minutes). Recovered aqueous phase was mixed with 1 volume of isopropanol and 1/10 volume of 3M sodium acetate, and stored at −80°C for 20 minutes for DNA precipitation. Samples were centrifuged (15000 rpm, 4°C, 20 minutes), supernatant discarded, washed with 70% ethanol, air-dried, and resuspended in nuclease-free water. The sample DNA extracts were further purified with DNeasy Blood & Tissue Kit, following manufacturer protocol (QIAGEN).

Nascent EU RNA-seq (neuRNA-seq) labeling and capture were done by using Click-iT Nascent RNA Capture Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Kc cells were incubated with 0.2 mM EU for 1 h and RNA was extracted with Trizol (Thermo Fisher Scientific). Next, RNA was chemically fragmented for 5 min at 70 °C with RNA Fragmentation Reagents (Thermo Fisher Scientific), followed by DNase I treatment (Roche). Then RNA was ethanol precipitated after Phenol:Chloroform (Thermo Fisher Scientific) purification. The Click-iT reaction was performed with 0.5 mM biotin azide using 5 µg of EU-RNA, and biotinylated RNA was captured with 12 μL T1 beads. The nascent EU-RNA was used to generate RNA-seq libraries with Ovation RNA-seq Systems 1–16 for Model Organisms (Nugen). Samples were sequenced on HiSeq2500 (Illumina) using 50 bp single-end sequencing at the NIDDK Genomics Core Facility.