Name: GSM7721060
Instrument: NextSeq 2000
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Three biological replicates of samples were extracted in three separate experiments (one per replicate). Each pellet (~ 40 O.D. * mL) was resuspended in 600 µL of lysis buffer (10 mM Tris pH 8.0, 1x cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche, cat. No. 11836170001), 50 mM NaCl, 3 µL of Ready Lyse lysozyme per mL) and incubated for 15 minutes at 30°C. Samples were placed on ice, then sonicated in the ice bath at 25% amplitude for 20 seconds (5 seconds on, 15 seconds off). The samples were clarified by centrifugation (16,000 r.c.f.,10 minutes, 4°C), and the liquid transferred into new microfuge tubes. To each sample, an equal volume of 2x IP buffer (200 mM Tris, pH 8.0, 600 mM NaCl, 4% (v/v) Triton X-100 with 2× protease inhibitors (Roche, cat. No. 11836170001) and 0.2 mg/ml BSA) was added and mixed by inversion. To preclear samples, 100 ?L of pre-washed (add 100ul of beads per sample in microfuge tube, place on magnetic stand, remove storage buffer, add 1 mL 1X IP buffer, mix by inversion, clear using magnetic stand, remove liquid, resuspend in 100 µL of 1x IP buffer per sample) protein G magnetic beads (NEB; lot 101325116) were added to each sample and incubated for 2 hours on a nutating platform at 4°C. Beads were cleared using a magnetic stand, and the liquid was transferred to new 2 mL microfuge tubes. To keep as input material, 100 µL of each sample was transferred to a new tube containing 400 µL of ChIP elution buffer (50 mM Tris, pH 7.0, 10 mM EDTA, 1% (w/v) SDS), mixed, and kept on ice until proteinase K (10 µL per sample; Fermentas, 50 mg/mL) digestion. To each ChIP sample, 8 µL of mCherry Monoclonal Antibody 16D7 (Invitrogen, M11217, lot #XC345714) was added and incubated for 2 hours on a nutating platform at 4°C. To capture the antibodies, 100 ?L of pre-washed (see above) protein G magnetic beads (NEB; lot 101325116) were added to each ChIP sample and incubated 2 hours on a nutating platform. Beads from the ChIP samples were washed, in series, with 1 mL of Buffer A (100 mM Tris pH 8.0, 250mM LiCl, 0.2% (v/v) Triton x-100, 1 mM EDTA), Buffer B (10 mM Tris pH 8.0, 500 mM NaCl, 0.1% (v/v) Triton x-100, 1 mM EDTA, sodium deoxycholate), Buffer C (10 mM Tris pH 8.0, 500 mM NaCl, 0.1% (v/v) Triton x-100, 1 mM EDTA), 1X IP buffer (100 mM Tris pH 8.0, 300 mM NaCl, 2% (v/v) Triton x-100, 1 mM EDTA) and 1X TE (10 mM Tris pH 7.0, 1 mM EDTA). Beads from the ChIP samples were resuspended in 500 ?L ChIP elution buffer. To recover the RNA, for each input and ChIP sample, 10 µL of RNase/DNase free proteinase K was added. After mixing by inversion, all tubes were incubated for 2 hr at 37°C. This was followed by phenol/chloroform extraction clean up (500 ?L of acidic 5:1 phenol/chloroform (VWR, E277) was added, mixed, and allowed to separate into layers at 16,000 r.c.f., 5 minutes, 4°C); the water layer was transferred to a new tube with 500 ?L of 24:1 chloroform/isoamyl alcohol, mixed, allowed to separate as before at 16,000 r.c.f., 5 minutes, 4°C); finally, the water layer was transferred to a new tube). Nucleic acids from the samples were precipitated by the addition of 1/25 volume of 5 M NaCl, 2 ?L of Glycoblue coprecipitant (Invitrogen), 1 volume of 100% (v/v) ethanol and 1 volume of 100% (v/v) isopropanol, mixed, and then incubated for 1 hour at 4°C, then > 2 hr (up to overnight) at -20°C. Nucleic acids were pelleted via centrifugation (16,000 r.c.f., 15 minutes, 4°C), washed once with ice-cold 95% (v/v) ethanol, and air dried. Pellets were resuspended in 83 ?L of 0.1X TE (1 mM Tris pH 7.0, 0.1 mM EDTA) and stored at -80°C. The DNA was removed from the samples 10 ?L of 10X Baseline-ZERO Buffer plus 5 ?L of Baseline-ZERO DNase (Lucigen) and 2 ?L of murine RNase Inhibitor (NEB), and incubated 37°C for 30 min. The RNA was collected/cleaned using the Zymo RNA Clean and Concentrate Kit-96 per kit directions (Zymo Research) and eluted with either 15 (extracts) or 20 ?L (inputs) of water. The concentration of RNA was determined with 3 ?L of each sample using QuantiFluor RNA System (Promega) and the provided standards. Input material obtained from Hfq-mCherry RNA ChIP was treated with NEBNext rRNA Depletion Kit (Bacteria) [NEB]. For each input, 11 µL of diluted material (0.15-0.44 µg) was used. Depletion performed per kit directions except that Zymo RNA Clean and Concentrate Kit-96 [Zymo Research, per kit directions (3000 r.c.f., 5 min centrifugation) and eluted with 11 ?L of water] was substituted for the post DNase I digestion bead clean-up in an attempt to preserve small RNAs that may be present. Library preparation was performed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) and either 5 ?L of rRNA depleted material (inputs) or 5 ?L of RNA (5 - 36 ng) obtained by immunoprecipitation (extracts). Samples were prepared per kit directions except that samples were fragmented for 1 min, the post-second strand synthesis bead clean-up was replaced with an Oligo Clean and Concentrate kit [Zymo Research, per kit directions (3000 r.c.f., 5 min centrifugation) and eluted with 25 ?L of water, and add 25 ?L of water prior to end prep reaction], diluted (1:20) NEBNext Unique Dual Index UMI Adaptors DNA Set 1 were used instead of the NEBNext Adaptor for Illumina, the post ligation bead binding involved 174 ?L of Axyprep beads (Axygen) and 68 ?L of isopropanol, and the NEBNext Primer Mix was used for the PCR amplification step. Pooled libraries were subjected to Illumina sequencing on a NextSeq platform with 38 x 37 bp paired end reads.