Illumina MiSeq paired end sequencing; Sequence Requirements for Cascade Function in a Type I-E CRISPR System: ChIP Library-seq
Sequence Requirements for Cascade Function in a Type I-E CRISPR System: ChIP Library-seq
1. Grew ON’s of strains to electroporate plasmids intoa. Binding assays: C-NC yggX* casA-F3 with pBAD33 (camR) 2. Made electrocomp cells and transformed in plasmids (ampR)(100ng into 50 µl of cells).3. Start overnights of each strain + plasmid a. Subculture 1/100 in 5 ml LB for 8 protospacer plasmids or 3 ml of LB for 13 protospacer plasmids.  Induce system with 0.2% arabinose. Grow to appropriate OD600 (typically 0.3-0.6).b. Mix 8 X 5 ml or 13 (pLC023-35) X3 ml of cultures together.c. Crosslink cells by adding formaldehyde to final concentration of 1% (~1 ml per 40 ml culture). Swirl flask to mix. Incubate for 20 min at room temperature.d.  Quench formaldehyde by adding 10 ml of 2.5 M glycine per 40 ml culture [final] = 0.5 M (typically).e. Spin cells at full speed 10 minutes in bench-top centrifuge and discard supernatant. f. Resuspend in 40 ml TBS (original culture volume). Spin and discard supernatant again.g. Resuspend in 1 ml TBS. Spin in microcentrifuge, max speed, 1 min. Discard supernatant. Cells can be frozen at this point. 1. Grew ON’s of strains to electroporate plasmids intoa. Binding assays: C-NC yggX* casA-F3 with pBAD33 (camR) 2. Made electrocomp cells and transformed in plasmids (ampR)(100ng into 50 µl of cells).3. Start overnights of each strain + plasmid a. Subculture 1/100 in 5 ml LB for 8 protospacer plasmids or 3 ml of LB for 13 protospacer plasmids.  Induce system with 0.2% arabinose. Grow to appropriate OD600 (typically 0.3-0.6).b. Mix 8 X 5 ml or 13 (pLC023-35) X3 ml of cultures together.c. Crosslink cells by adding formaldehyde to final concentration of 1% (~1 ml per 40 ml culture). Swirl flask to mix. Incubate for 20 min at room temperature.d.  Quench formaldehyde by adding 10 ml of 2.5 M glycine per 40 ml culture [final] = 0.5 M (typically).e. Spin cells at full speed 10 minutes in bench-top centrifuge and discard supernatant. f. Resuspend in 40 ml TBS (original culture volume). Spin and discard supernatant again.g. Resuspend in 1 ml TBS. Spin in microcentrifuge, max speed, 1 min. Discard supernatant. Cells can be frozen at this point. Immunoprecipitation (with protein A beads) Do X2 for IP to get enough DNA1. Transfer 20 ul to fresh tube for Input DNA control2. Add 25 μl protein A beads (from 50% slurry in TBS)3. Add appropriate amount of antibody [2 anti-FLAG) 4. Incubate for 90 min at room temperature on rotisserie rotator.5. Spin IP mix for 1 minute in microcentrifuge at 4,000 rpm. Remove supernatant by aspiration. 6. Resuspend in 700 μl FA lysis buffer and transfer to Spin-X column filter. Incubate for 3 min at room temperature on rotisserie rotator. Spin (as before) and discard supernatant7. Wash with 750 μl of each of the below buffers by incubating for 3 min at room temperature on rotisserie rotator, spinning for 1 minute in microcentrifuge at 4,000 rpm and discarding supernatant:FA lysis bufferFA lysis buffer (again)FA lysis buffer + 500 mM NaCl, ChIP wash buffer, TE (10 mM Tris pH 8.0, 1 mM EDTA). 8. Place Spin-X filter in fresh dolphin-nosed tube. 9. Resuspend beads in 100 μl ChIP elution buffer and incubate for 10 min at 65 °C with occasional gentle mixing.10. Spin for 1 minute in microcentrifuge at 4,000 rpm. Discard filter.11. Transfer to normal 1.5 ml tube and incubate 10 min at 100 °C (Bring to rolling boil and then put at 380) to Decrosslink. Include 20 ul input sample (add 80 microliters ChIP elution buffer to total 100). 12. Clean up ChIP and Input DNA with Zymo PCR purification kit (use 2V of binding buffer), elute w/ 200 ul H2O for IC and 75 ul H2O per IP for a total of 150 ul H2O.6) Check concentrations with Qubita) 5 µL into 195 µL for ICb) 5 µL into 195 µL for IP  Making Mi-seq libraryPCR• 5.48 ng of Chromatin from ChIP• 50 µL Failsafe Rxno 0.5 Failsafe, 0.5 fwd, 0.5 rev, Rest of 23.5 with dH2O, 25 Buffer “C” Ultramers: 1:10 8567 and 1:100 8556-8566 & 8537• 98° 2 min, (95° 30 sec, 72° 30 sec., 72° 30 sec. 25 cycles)0.8X Ampure bead clean-up, elute with 11µL dH2O, Qubit 1 microliter