1. Collect worms in a 50ml tube. Spin and wait until worms are collected at the bottom. Transfer sample to a 15ml tube and wash with M9 until clean.

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1 Worm Collection 1. Collect worms in a 50ml tube. Spin and wait until worms are collected at the bottom. Transfer sample to a 15ml tube and wash with M9 until clean. 2. Transfer sample to a 50ml conical. Add M9 up to 47.5ml mark. Add 2.8ml of formaldehyde. Rotate at RT for 28. Spin down cross-linked sample and remove supernatant. 3. Make FA Buffer during step 2. 25ml of RT FA Buffer + 1 Roche Complete tablet (Cat# , Protease Inhibitor Cocktail tablets). 4. After 28 incubation, spin and remove supernatant. Wash with 1M Tris ph7.5. Spin and remove supernatant. 5. Wash 2 times with M9, spin, and remove supernatant. 6. Transfer to a 15ml conical. Spin and remove supernatant. 7. Wash with 12-15mls of FA Buffer. Spin and remove as much supernatant as possible. Worms may stick to 15 ml conical so add just a drop of FA buffer to release them from the sides of the tube. Want ul of pelleted worms. 8. Flash freeze in liquid nitrogen and store at -80 C. Day 1 Chromatin Immunoprecipitation 1. Add 1 Roche Complete tablet, 25ul 1M DTT, 125ul of 100mM PMSF to 25mL of chilled FA Buffer (for 4 samples). Rotate at RT to dissolve pellet. Once dissolved, keep on ice. 2. During step 1 rotation, thaw and always keep samples on ice. After thawed, add FA buffer to 3ml mark. 3. Turn on sonicator and check program. Make sure the dial is set to Microtip. Press Program twice in order to see last program used. Total Sonication ON time- 2:40 (~16 cycles = 17 total to achieve bp fragment) ON Time/Cycle- 0:10 OFF Time/Cycle- 1:00

2 4. Prepare ethanol ice bath by filling 500ml plastic beaker with ice. Add 1:1 of 95% ethanol and water to ice. Stir to mix. 5. Clean microtip by washing with 95% ethanol and then di-water. Clean microtip for 1 sonication cycle using a conical containing di-water. 6. Place sample in chamber and secure the conical with the clamp, keeping microtip 2-3mm from the bottom and away from the sides of the conical. Keep sample submerged in ethanol/ice bath as much as possible. Close and tape door. Press start. Use a folded kim wipe to help secure the clamp to the conical tube. Make sure the conical stays within the layer of ice. After first round of sonication, make sure the sample is not frozen. 7. Rinse microtip with water, ethanol, water. Clean microtip for 1 sonication cycle using a conical containing di-water. 8. Repeat 6 & 7 until all samples are sonicated. 9. Clean microtip for 3 sonication cycles and turn off sonicator. 10. Put 1.5ml sample each in to two 2ml tubes. Spin at 13,000 x g for 15 at 4 C. Label tube with strain, replicate number, date collected and sonicated, and protein concentration. 11. Transfer supernatant in to a 15 ml conical tube avoiding pellet. 12. Determine protein concentration by Bradford assay. a) BSA Standard (2mg/ml): 8- standards from 2mg/ml 0 mg/ml (2, 1, 0.5, 0.25, 0.125, , , 0). 20ul of appropriate BSA standard + 980ul of Bradford reagent 2mg/ml standard has 20ul of BSA standard. Do serial 1:2 dilutions for 1mg/ml standards. 0mg/ml has 20ul of water. b) Sample: 2- sample tubes of 1:10 and 1:20 dilutions. 20ul of appropriate dilution +980 ul of Bradford reagent. 1.5ul of sample, in total volume of 30ul for 1:20 dilution and serial dilute to make 1:40. Transfer to cuvette and spec. Be sure to change the units to mg/ml, the number of standards to 8, and change standard protein concentration to your standard concentration. Determine the protein concentration.

3 Multiply protein concentration determined by the spec and dilution factor. Average the concentration. 13. Add extract corresponding to ~2.2mg protein to a 1.5ml tube and bring the volume to 400ul with chilled FA Buffer+ Protease Inhibitors. Then add 1:20 (v:v) 20% sarkosyl (20ul) and spin at 13,000x g for 5 minutes at 4 C. If preparing an IgG control, use 4.4mg protein in a total volume of 800ul and 40ul sarkosyl. Flash freeze remainder and store at -80 C. 14. Transfer the supernatant to a new tube. Remove up to 40uL of the material and store it at -20 C until the following day, when it will be used to prepare input DNA. 15. Add appropriate amount of antibody (7.5ug of GFP antibody from Tony Hyman s lab=0.5ul) and rotate overnight at 4 C (16-20h). If preparing the IgG control, split the remaining extract between two tubes (~2.2mg each), one for the ChIP antibody (GFP antibody from Tony Hyman s Lab) and one for the control goat IgG antibody* (R&D Systems, Cat. #AB-108-C). 7.5ug of each antibody. 0.5ul is difficult to pipette so if preparing multiple samples, make a 1:10 dilution master with FA, then add 5 ul to ChIP sample. For RPC-1 IP, Use 4ug RPC-1 antibody (from Jason Leib s lab) per 4mg protein( in 800ul volume), incubate with 40ul protein A sepharose beads (GE Healthcare Cat. # ), and still elute twice with 150ul elution buffer. Day 2 1. Thaw the input samples from the previous day and add 2uL 10mg/mL RNase A. Digest at RT for 2 hours. 2. While the input samples are thawing, begin washing beads. Take 35uL (~20ul of actual beads) of protein G sepharose beads (GE Healthcare Cat. # ) per ChIP/IgG sample and wash 4 times with 1mL FA buffer. Spin at 2500x g (rcf) for 2 minutes to collect the beads. Wash scissors with ethanol and cut pipette tip when pipetting the beads. Can use the FA buffer + protease inhibitors from the day before. Keep FA buffer on ice. Compare the volume of spun down beads with a 1.5 ml tube containing 20ul of water to ensure there is enough beads. Use vaccum and 1-10ul tips to remove supernatant. 3. Add washed bead slurry to each ChIP sample and continue to rotate at 4 C for 2 hours. At 4 C, quickly spin ChIP samples to collect sample to the bottom of the tube. Add 50 ul of ChIP sample to beads and resuspend by pipetting. Transfer the beads to the rest of the ChIP sample. Keep on ice.

4 Option2: transfer all 400ul ChIP sample to the beads 4. After the 2 hour RNase A treatment (Step 1 for the input samples), add 260uL elution buffer (or enough to bring volume up to 300uL), then add 4uL of 10mg/mL Proteinase K (Roche cat # or ) and put the input sample at 55 C for 2-4 hours. If there is precipitate in the elution buffer, put at 42 C to return back to solution. We are currently using Proteinase K (Roche cat# ) 5. After the 2 hour bead incubation (Step 3 for the ChIP samples), wash beads at RT by adding 1 ml of each of the following buffers and incubating for the specified time on a rotator. Collect beads by spinning for 2 minutes at 2500x g: 2 times RT FA buffer (150mM) for 5 minutes 1 time FA-1M NaCl for 5 minutes. After this wash, transfer beads to new tubes with the next wash buffer. 1 time FA-500mM NaCl for 10 minutes 1 time TEL buffer for 10 minutes 2 times TE for 5 minutes **OPTIONAL: For IP-Western, resuspend beads in 50uL of 2x sample buffer. 6. To elute the immunocomplexes, add 150uL elution buffer and place the tube in a 65 C heat block for 15 minutes. Vortex briefly every 5 minutes. 7. Spin down the beads at 2500x g for 2 minutes and transfer the supernatant to a new tube. Use western loading tips to remove about 150ul of supernatant. After the first elution, leave some supernatant behind so you don t disturb the beads. 8. Repeat elution and combine supernatants. Try to remove as much supernatant as possible. 9. Add 2uL of 10mg/mL Proteinase K to each ChIP sample. Incubate for 1-2 hours at 55 C. 10. Transfer all input and ChIP samples to 65 C for hours to reverse crosslinks. Day 3 1. Purify the DNA with Qiaquick PCR purification kit (Qiagen Cat ). Elute with 34uL H2O. Use a 2ml tube when mixing the PB buffer and sample. Run 5uL of the input on a 1.5% agarose gel to check the extent of shearing. Most of the DNA fragments should be bp. Elute with 50uL if you will also use the ChIP DNA for qpcr. It is also ok to elute with EB instead of water.

5 Freeze samples after this or any other DAY3 Qiagen purification kits if you can t finish the library preparation in one day. Library Preparation for Illumina ChIP-Seq STEP 1: End repair using End-It DNA End Repair Kit from Epicentre, Cat# ER81050 (50 reactions). 2. Combine the following components in a microfuge tube: 1-34 ul ChIP DNA to be end-repaired (200ng if it is Input DNA) 5uL 10X End-Repair buffer 5uL 2.5mM dntp mix 5uL 10mM ATP xul sterile water to bring reaction to 49uL 1uL End-Repair enzyme mix 50uL Total 3. Incubate at room temperature for 45 minutes. 4. Purify using QIAquick PCR Purification Kit and protocol, eluting in 34uL of EB. STEP 2: Addition of A base to 3 Ends (Use Klenow 3 5 exo- from NEB Cat#M0212s or M0212L) 5. Combine and mix the following components in a PCR plate DNA from STEP 1 34uL Klenow Buffer (NEB Buffer 2) 5uL 1mM datp 10uL Klenow fragment (3 5 exo-) 1uL 50uL total To make 1mM datp using NEB 100mM ATP, add 10uL of 100mM datp to 990uL Qiagen Buffer EB; then make 100uL aliquots and freeze at -20 C. 6. Incubate for 30 minutes in a 37 C water bath. 7. Purify using QIAquick MinElute column using the MinElute PCR Purification Kit (Qiagen Cat ) and protocol. Elute in 12uL EB. STEP 3: Adapter Ligation Use LigaFast from Promega Cat#M8221 (30 reactions) or M8225 (150 reactions). ModENCODE uses 4 barcoded adapter mixes/flow cell :1, 6, 8, 9 8. Combine and mix the following components in a microfuge tube. DNA from STEP 2 12uL 2x DNA ligase buffer 15uL

6 Adapter oligo mix (~30ng/ul) DNA ligase 1uL 2uL 30uL total 9. Incubate reaction for 15 minutes at room temperature. 10. Purify using the MinElute PCR Purification Kit and protocol. Elute in 20uL EB. 11. Plug in E-gel apparatus. Place E-gel in apparatus and make sure the apparatus light is red. Label E-gel. 12. Remove comb and load 20ul of ladder and sample, leaving a blank well between each one. Fill the blank wells with 20ul of water. Press 30 button and make sure the light turns green. When the gel is done running, the light will return to red. The gel is actually done running at about 17minutes, so check your gel in17 minutes and do not run your sample off the gel. You can also use a poured 2% agarose TAE gel. Take a picture of the samples after the E-gel run is complete. When use E-gel, you can skip the loading dye for both your samples or DNA ladder Dilute 100bp DNA ladder to the concentration of 7.5ng/ul 13. Using a clean scalpel every time, cut a gel slice for each sample corresponding DNA in the bp range. Do NOT include any DNA from an adapter-adapter band migrating at ~120 bp. Do not cut over 400bp. The intended ligation product may not be visible at this stage for chip samples. Label the front and back of the E-gel with the area you intend to cut. Take a picture of the E-gel before and after it is cut. 14. Purify the DNA from the agarose slice using a QIAquick MinElute gel extraction kit (Qiagen Cat ). Dissolve gel slice at 37 C in 500ul of Buffer QG. Perform the wash step with 500uL Buffer QG prior to the Buffer PE wash step. Elute in 23uL EB. STEP 4: PCR and size selection Illumina PCR primer 1.1 Illumina PCR primer 2.1 See Illumina PCR Primers below 15. Combine and mix the following components in a PCR tube: DNA from STEP 3 Phusion PCR master mix (NEB # F-531S or M0531L ) PCR primer 1.1 (14.3pmoles/uL stock) PCR primer 2.1 (14.3pmoles/uL stock) 23uL 25uL 1 ul 1 ul

7 Total reaction volume 50uL Amplify using the following PCR protocol: C, 30 sec C, 10 sec C, 30 sec C, 30 sec Cycle Steps C, 5 min 7. 4 C Hold 16. Purify using the MinElute PCR Purification Kit and protocol. Elute in 20µl EB. 17. Plug in E-gel apparatus. Place E-gel in apparatus and make sure the apparatus light is red. Label E-gel. 18. Remove comb and load 20ul of ladder and sample, leaving a blank well between each one. Fill the blank wells with 20ul of water. Press 30 button and make sure the light turns green. When the gel is done running, the light will return to red. The gel is actually done running at about 17minutes, so check your gel in 17 minutes and do not run your sample off the gel. You can also use a poured 2% agarose TAE gel. Take a picture of the samples after the E-gel run is complete. When use E-gel, you can skip the loading dye for both your samples or DNA ladder Dilute 100bp DNA ladder to the concentration of 7.5ng/ul 19. Using a clean scalpel every time, cut a gel slice for each sample corresponding DNA in the bp range. Do NOT include any DNA from an adapter-adapter band migrating at ~120 bp. Do not cut over 400bp. Label the front and back of the E-gel with the area you intend to cut. Take a picture of the E-gel before and after it is cut. 20. Purify the DNA from the agarose slice using a QIAquick MinElute gel extraction kit (Qiagen Cat ). Dissolve gel slice at 37 C in 500ul of Buffer QG. Perform the wash step with 500uL Buffer QG prior to the Buffer PE wash step. Elute in 15uL EB. 21. Measure concentration of DNA in each sample using a nanodrop machine. Use 2uL for each sample. Record the 260/ For one multiplex, mix 4 samples at ratio 1:1:1:1 in one tube. Measure concentration of the multiplex using a nanodrop machine. Use 2uL for each sample. Record the 260/280.

8 Make sure that the 4 samples that you are mixing have different adaptors. The minimum total volume is 17ul (2ul to spec, 5ul for Keck bioanalyzer, 10ul to ship). To best achieve this minimum volume use 5-6ul of the least concentrated sample, and then the same amount of DNA for the 3 remaining samples. Reagents For Extract Preparation, Collection of the Immunocomplexes, and Washes. Filter sterilize FA and TEL Buffers and store at 4 C (Good for at least 6 months). 1) FA Buffer (150mM NaCl): 50mM HEPES/KOH ph7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150mM NaCl Recipe (500 ml): 1M HEPES-KOH, ph ml 0.5M EDTA, ph ml Triton-x ml 5% DOC (deoxycholic acid) 10 ml 5M NaCl 15 ml Sterile water 444 ml 2)FA-1M NaCl Buffer: 50mM HEPES/KOH ph7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1M NaCl Recipe (500 ml): 1M HEPES-KOH, ph ml 0.5M EDTA, ph ml Triton-x ml 5% DOC (deoxycholic acid) 10 ml 5M NaCl 100 ml Sterile water 359 ml 3)FA-500mM NaCl Buffer: 50mM HEPES/KOH ph7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 500mM NaCl Recipe (500 ml): 1M HEPES-KOH, ph ml 0.5M EDTA, ph ml Triton-x ml 5% DOC (deoxycholic acid) 10 ml

9 5M NaCl Sterile water 50 ml 409 ml 4)TEL Buffer: 0.25M LiCl, 1% NP40, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl, ph8.0 Recipe (250ml): 5M LiCl 12.5 ml NP ml 5% DOC (deoxycholic acid) 50 ml 0.5M EDTA, ph ml 1M Tris-HCl, ph ml Sterile water 182 ml 5) Elution Buffer: 1% SDS in TE with 250mM NaCl Recipe (50 ml): 20% SDS 2.5 ml 5M NaCl 2.5 ml TE 45 ml 6) 20% Sarkosyl Solution: Recipe (100 ml): N-lauroyl-sarcosine, sodium salt Sterile water 20g 100 ml

10 Illumina Adapters (HPLC purified) Oligonucleotide sequences for generation of the four barcoded adapters Oligo name Barcode Modification Sequence (5' 3') MPLEXA1F GTAT None ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAT MPLEXA1R GTAT 5' phosphate TACAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG MPLEXA6F CATT None ACACTCTTTCCCTACACGACGCTCTTCCGATCTCATT MPLEXA6R CATT 5' phosphate ATGAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG MPLEXA8F ACGT None ACACTCTTTCCCTACACGACGCTCTTCCGATCTACGT MPLEXA8R ACGT 5' phosphate CGTAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG MPLEXA9F TGCT None ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCT MPLEXA9R TGCT 5' phosphate GCAAGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG Lefrançois et al. BMC Genomics :37 doi: / Instructions on how to prepare them: BMC Genomics 21 January :37 Lefrancois et al. Illumina PCR Primers (HPLC purified) PCR 1.1 5' AATGATACGGCGACCACCGACACTCTTTCCCTACACGACGCTCTTCCGATCT PCR 2.1 5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT