GENERATION OF HIC LIBRARY

GENERATION OF HIC LIBRARY Gel checking points during one HiC experiment ------ % gel Step Checking list 1 0.8 After cells are lysed Pellet degradation check 2 0.8 After template generation 1 template quality 2 template concentration (for sonication) 3 2.0 Efficiency check PCR 1 ligation efficiency 2 biotinylation efficiency 4 2.0 Sonication 1 sonicated DNA size range 2 concentration (for MiniElute, datp) 5 2.0 AMPure size selection 1 DNA size range 2 concentration (for pull-down) 6 2.0 PE PCR cycle titration 1 titration curve 2 DNA yield per reaction 3 - DNA range (for AMPure purification) 7 2.0 Nhe1 digestion 1 - % of dangling ends per library 8 2.0 After AMPure purification 1 final library concentration 2 DNA size range HiC protocol for John Stam Collaboration - Ye Page 1

2. Cell lysis and chromatin digestion--- use one batch of cells (~ 20M cells) 2.1) To lyse the cells, 1 ml lysis buffer (500 µl 10 mm Tris-HCl ph8.0, 10 mm NaCl, 0.2% Igepal CA630) is mixed with 100 µl protease inhibitors and added to one batch of cells (~ 20-25 million cells). 2.2) Cell suspension is incubated on ice > 15 minutes 2.3) Cells are lysed with a homogenizer with a pestle A, douncing 30 times on ice; then incubate on ice > 1 min and then give another 30 strokes 2.4) Transfer the lysis to a 1.7ml tube and centrifuged for 5 minutes at 5000 rpm at RT, discard the supernatant 2.5) Wash each pellet twice with 500 µl of ice cold 1x NEBuffer 2 2.6) Resuspend each pellet in 1x NEBuffer 2 in a total volume of µl and aliquot 50 µl per tube --- save about 5µl lysate for pellet degradation check --- At this step, save half volume at -80 C for 3C_seq 2.7) Add 312 µl 1x NEBuffer 2 per tube 2.8) To remove the proteins that are not directly crosslinked to the DNA, 38 µl 1% SDS is added per tube. Resuspend carefully and incubate the mixture at 65 C x 10 minutes exactly, then place on ice immediately 2.9) Add 44 µl 10% Triton X-100 to quench SDS, mix carefully and avoid making bubbles 2.10) Chromatin is subsequently digested overnight at 37 C by adding 400 Units HindIII. Rotate tubes while incubating. Cell pellet degradation check ---- a) Mix 5µl cell lysate, 10µl of 1xNEBuffer 2 and 5µl of 10mg/ml proteinase K b) Incubate at 65 C x 30min c) Check the stickiness of the product by pipetting or load everything very carefully on 0.8% gel and check the integrity of the DNA HiC protocol for John Stam Collaboration - Ye Page 2

3. Marking of DNA ends and blunt-end ligation 3.1) All tubes with digested chromatin are spun shortly at 5000 rpm and are put on ice 3.2) The DNA ends are filled in and marked with biotin by adding 60 µl fill-in mix (see table below) to HIC tubes Fill-in mix (add in this order) Per reaction Water 2.0 µl 10x NEBuffer 2 6.0 µl 10 mm datp 1.5 µl 10 mm dgtp 1.5 µl 10 mm dttp 1.5 µl 0.4 mm biotin-14-dctp 37.5 µl 5U/ µl Klenow 10.0 µl 3.3) Mix carefully and incubate at 37 C x 4 hours on a rotator. Place the tubes on ice directly afterwards 3.4) To inactivate the enzymes, 96 µl 10% SDS is added to each tube and incubate at 65 C x 30 minutes exactly and placed on ice immediately afterwards 3.5) Pre-chill 15 ml tubes on ice, add 7.58 ml ligation mix (see table below) to each tube Ligation mix Per reaction Water 5,838 µl 10x ligation buffer (Dekker lab s) 758 µl 10% Triton X-100 820 µl 10 mg/ml BSA 82 µl 100 mm ATP 82 µl 3.6) Transfer each digested chromatin mixture to a corresponding 15 ml tube, add T4 DNA ligase to each tube and mix well by inverting the tubes and --- for normal 3C ligation, 10 µl 1U/µl T4 DNA ligase is added (skip this step) --- for blunt-end Hi-C ligation, 50 µl 1U/µl T4 DNA ligase is added 3.7) Incubate all tubes at 16 C x 4 hours (some samples may need overnight ligation) HiC protocol for John Stam Collaboration - Ye Page 3

4. DNA purification 4.1) Crosslinks are reversed and protein is degraded by adding 50 µl 10 mg/ml proteinase K per tube and incubating the tubes at 65 C for 4~5 hours 4.2) Add another proteinase K and then continue incubating at 65 C overnight 4.3) Cool the reaction mixtures to RT and transfer them to 50 ml conical 4.4) Add equal volume of phenol: chloroform (1:1) per tube and vortex 1 min 4.5) Use 50ml phase lock tubes to purify DNA 1,500 x g x 5 min 4.6) Transfer the aqueous phases to new 50 ml conical tubes, add equal volume of phenol: chloroform (1:1) per tube, vortex for 1 min 4.7) Use the same phase lock tubes to extract the DNA 4.8) Transfer the aqueous phases the 250 ml centrifugation bottles 4.9) For DNA precipitation, add 10% volumes of 3M Na-acetate per tube and mix well before adding 2.5 volumes of ice-cold 100% ethanol. Mix properly by inverting the bottles several times and place the tubes at -80 C > 1h 4.10) Spin at 4 C x 20 minutes at 12,000 x g, discard the supernatant 4.11) Dissolve each DNA pellet in 2 x 2 ml of 1x TE and then transfer the DNA mixture to a 15 ml conical 4.12) Add equal amount of phenol ph8.0: chloroform (1:1) to each tube, vortex for 1 min and extract DNA using the 15ml phase lock tubes 1,500 x g x 5 min 4.13) Repeat step 4.9) once more 4.14) Transfer the upper phases to the 35 ml centrifugation tubes 4.15) Add 10% volumes of 3M Na-Acetate and 2.5 volumes of ice cold 100% ethanol per tube, mix by inverting the tubes several times and place the tubes at - 80 C for at least 1 h 4.16) Spin at 18,000 x g x 20 minutes at 4 C, then discard the supernatant 4.17) Air dry the pellets at RT 4.18) Dissolve each pellet in 3 ml + 2 ml of 1X TE buffer 4.19) Remove the excessive salt from the sample by using AMICON Ultra Centrifugal Filter Unit 15 ml 30K as follows -- 4.19.1) Transfer DNA sample to the Amicon ultra filter devices 4.19.2) Spin at 4000 rpm x 15 min, discard the flow-through 4.19.3) Add 5 ml of 1X TE buffer and spin at 4000 rpm x 15 mim 4.20) Transfer the collected sample to a 1.7ml centrifuge tube using a pipette tip 4.21) Any purified RNA is degraded by adding 1µl of 10 mg/ml RNAse A to each HiC and incubating the tubes at 37 C x 30 minutes. (or add 1/10 volume of 10 mg/ml RNase A per sample) HiC protocol for John Stam Collaboration - Ye Page 4

4.22) Store at -20 C 5. Biotinylation and ligation efficiency check by PCR 5.1) To check the quality and quantity of the libraries, aliquots from both 3C and Hi-C libraries are run on a 0.8% agarose gel. Typically, run 5 and 10 µl of a 1:10 dilution of the libraries 5.2) Hi-C marking and Hi-C ligation efficiency is verified by a PCR digest assay. Successful fill-in and ligation of HindIII sites (AAGCTT) should create sites for the restriction enzyme NheI (GCTAGC) Notes --- 1) Normally add 80ng of DNA into a 30µl PCR reaction 2) Always run duplicate for each PCR reaction 3) The primers for quantifications are including --- Human_HindIII AHF 62 + AHF 64 Mouse_HindIII mgapdh 3 + mgapdh 4 Fly_HindIII NB 160 + NB 161 --- A ligation product formed from two nearby restriction fragments is amplified using 3C PCR and the PCR products are subsequently digested with HindIII, NheI or both. After running the samples on a 2% gel, the relative number of 3C and Hi-C ligation events can be quantified HiC protocol for John Stam Collaboration - Ye Page 5

6. Removal of Biotin from the un-ligated ends 6.1) Multiply the 5 µg of DNA/ reaction to obtain the desired amount of HiC HiC library 5 µg 10 mg/ml BSA 0.5 µl 10X NEBuffer 2 5 µl 2.5 mm datp 0.5 µl 2.5 mm dgtp 0.5 µl 3,000 U/ml T4 DNA polymerase 5 µl (15 units) Water make up to a total 50 µl volume --- 20 C x 4 h followed by 75 C x 20 min in the PCR blocks 6.2) Pool the reactions together and transfer to Amicon columns 6.3) Spin at full speed at soft setting for 1 minute intervals until the remaining volume is < 100 µl --- In general, after 2 ~ 3 spins, the volume will be around 60 ~ 80 µl 6.4) Transfer the DNA left inside the column to a clean 1.7 ml tube with a pipette tip 6.5) Bring the total volume of HiC to ~ 105 µl with water HiC protocol for John Stam Collaboration - Ye Page 6

7. DNA sonication, size fractionation, DNA repairing, MiniElute purification and A-tailing 7.1) Shear the DNA to a size of 100 300 bp using a Covaris apparatus (main DNA ~ 200bp) 7.1-1) Fill the tank with fresh de-ionized water to top of blue tape fill line. 7.1-2) Turn on machine and start Covaris software. 7.1-3) De-gas water for at least 30 minutes 7.1-4) Set the chiller to 1-4 C (the temperature software display should settle near 7 C) 7.1-5) Load 105 ul Hi-C sample into a microtube 7.1-6) Keeping the cap on the tube, using a tapered pipette tip, transfer 105 µl of DNA sample by inserting the pipette tip through the pre-split septa. With the pipette tip approximately half way down the interior of the tube and alongside the interior wall, slowly discharge the fluid into the tube --- Be careful not to introduce a bubble into the bottom of the tube. This may happen if the sample is loaded too quickly --- It is important NOT to have any bubbles at the bottom of the tubes. Inspect every tube by raising the holder and check for bubbles in the tubes. Briefly and gently centrifuge to remove any bubbles 7.1-7) Carefully load microtube into the Covaris approved S2 holder, keeping tubes in a vertical orientation --- Take care not to bounce the rack or holder and carefully insert into the instrument. Make sure the water level is up to the level of the tube 7.1-8) Program the software with the desired settings (click configure and then return to main panel when finished entering parameters) 7.1-9) Initiate and Run the following process For all: Duty Cycle 10% Intensity 5 Cycles per Burst 200 Set Mode Frequency sweeping Continuous degassing Process time: 60 sec per process 7.1-10) Run the 60 sec process 2.5 ~ 3 times (2 min 30 sec ~ 3 min total), remove 2 µl and check on the 2% gel to determine whether a longer sonication is needed 7.1-11) Sonicate for additional time if necessary to achieve the desired size 7.1-12) To transfer liquid back to microcentrifuge tubes, use a long thin tip (gel-loading type) to remove liquid from covaris tube and then briefly spin the covaris tube and transfer the remaining to the same microcentrifuge tube HiC protocol for John Stam Collaboration - Ye Page 7

7.3) Use AMPure XP (pre-warm at RT) to select DNA with size range from 150 350 bp (when using 0.9X and 1.1X combination, you will get tighter band around 200bp, but you also lose tons of DNA between 200 300bp) 7.3-1) Pool all same reactions together 7.3-2) Add 2X or 4X volumes of EB buffer (volume A) to each sample 7.3-3) Add 0.8X volume of AMPure mixture (volume B) to volume A (from 7.3-2) to each sample, distributing the sample into 2 tubes if needed 7.3-4) Pipet to mix well, then incubate at RT x 10 min 7.3-5) Meanwhile, prepare 1.3X AMPure mix as follows --- 7.3-5A) Add the 2X volume of AMPure mixture as volume B to each clean tube 7.3-5B) Reclaim the beads against the MPC x 5 min and then discard all supernatant 7.3-5C) Resuspend each collected beads in the following volume of AMPure mixture --- (volume A) x 1.3 (volume B) 7.3-6) Reclaim the tubes from step 7.3-4) against the MPC x 5 min and then transfer all supernatant to the 1.3X AMPure mix from step 7.3-5C) 7.3-7) Mix well and incubate at RT x 10 min 7.3-8) Reclaim the beads against the MPC x 5 min and then remove all supernatant (save it) 7.3-9) Wash each beads from step 7.3-6) (0.8X) and 7.3-8) (1.3X) with 1ml of 70% Ethanol while the magnetic beads remained against the MPC without disturbing the separated beads --- x 2 times 7.3-10) Air dry the beads 7.3-11) Resuspend each pellet in 52 µl of TLE (EB, water) and incubate at RT x 10 min 7.3-12) Reclaim the beads against the MPC x 5 min 7.3-13) Transfer all supernatant to clean tubes keep 0.8X and 1.3X portions separately 7.3-14) Load 2 ~ 3 µl of each DNA on 2% TBE gel (along with 10 µl of supernatant) and run to check whether a desirable DNA range is achieved and measure the DNA concentration 7.3-15) If necessary adjust the combination of ratios of AMPure XP mix to sample and perform another size selection with the sample that is already size-selected HiC protocol for John Stam Collaboration - Ye Page 8

7.4) Repair DNA ends as follows Size-fractionated DNA 50.5 µl (adjust volume with Milli-Q water) 10X ligation buffer (NEB) 7 µl 2.5 mm dntp mix 7 µl T4 DNA polymerase (3U/ µl) 2.5 µl T4 polynucleotide kinase (10U/ µl) 2.5 µl Klenow DNA polymerase (5U/ µl) 0.5 µl --- 20 C x 30 min in the PCR blocks (program: 20ºC) to repair the DNA ends 7.5) Purify the DNA using QIAGEN MinElute columns (5 µg DNA per column) and elute DNA with twice of 17 µl of TLE buffer (10 mm Tris Cl, ph8.0, 0.1 mm EDTA) 7.5-1) Pre-warm elute buffer (EB, TLE or water) at 65 C 7.5-2) Pool the same reactions together, then add 5 volumes of Buffer PB and mix well 7.5-3) Transfer to MiniElute columns (5 µg DNA per column) and spin at 1,000 x g x 1 min, 2,000 x g x 1 min and 18,000 x g x 1 min 7.5-4) Wash each column with 750 µl of Buffer PE, spin at 1,000 x g x 1 min 7.5-5) Transfer the columns to new 1.7ml tubes, spin at 18,000 x g x 2 min 7.5-6) Transfer the columns to new 1.7ml tubes 7.5-7) Add 17 µl of pre-warmed elute buffer to each membrane of the column, incubate at RT x 3 min and then spin at 18,000 x g x 1 min 7.5-8) Repeat elution step once with another 17 µl of pre-warmed elute buffer 7.5-9) Combine the two elutes together 7.6) Perform the following reaction for each elute purified DNA (one elute) 32 µl 10X NEBuffer 2 5 µl 1 mm datp 10 µl Klenow (exo-) (5U/ µl) 3 µl (PCR program 37C65C to combine 7.5) and 7.6)) --- 37 C x 30 min in the PCR block to attach datp to the 3 end of the end-repaired DNA 7.7) Incubate at 65 C x 20 min to inactivate Klenow (exo-) and then place the DNA on ice right away HiC protocol for John Stam Collaboration - Ye Page 9

8. Biotin pull-down --- all subsequent steps are performed in DNA LoBind tubes (Eppendorf) 8.1) Vortex the streptavidin beads and then transfer corresponding amount of resuspended beads to each 1.7 ml tube (100 µl of beads ~ 20 µg of DNA) (--- The binding capacities of 1 mg of Dynabeads = ~20 µg dsdna) (the beads conc = 10 mg/ml) --- Every 1µg DNA requires 5µl of streptavidin beads 8.2) Wash each beads with 200 µl of TWB and incubate at RT x 3 min with rotation 8.3) Reclaim the beads against the MPC x 1 min and then remove all supernatant 8.4) Repeat wash step 8.2) and 8.3) once --- After this and for all subsequent incubation and washes of streptavidin beads, the beads are reclaimed by putting the tubes against the (MPC) x 1 min, then remove ALL supernatant (wash reclaim remove resuspend transfer) 8.5) Add TLE to each DNA to make a total volume 200 µl 8.6) Resuspend the reclaimed beads with 200 µl of 2X Binding Buffer (BB) and 200 µl of HiC DNA (so the final BB will be 1X), then incubate the mixture at RT x 15 min with rotation to allow the biotion labeled HiC DNA to bind to the streptavidin beads 8.7) Reclaim the beads against the MPC x 1 min, remove the supernatant 8.8) Resuspend the beads in 200 µl of 1X BB and transfer to a new tube 8.9) Reclaim against the MPC x 1 min, remove the supernatant 8.10) Wash the beads with 100 µl of 1X ligation buffer (5X T4 DNA ligation buffer from Invitrogen), and transfer to a new tube 8.11) Reclaim the beads again the MPC x 1 min, then remove the supernatant 8.12) Finally resuspend the beads in 38.75 µl (for ligation purpose) of 1X ligation buffer (5X T4 DNA ligation buffer from Invitrogen) HiC protocol for John Stam Collaboration - Ye Page 10

9. Paired end sequencing --- all subsequent steps are performed in DNA LoBind tubes (Eppendorf) 9.1) Set up one linkering reaction as follows and incubate at RT x 2h (can be stored at -20 C) Illumina paired end adapter 6 µl HiC DNA in 1X ligation buffer (Additional) 5X T4 ligation Buffer 38.75 µl (contains 7.75 µl of 5X T4 ligase buffer) 2.25 µl (total 10 µl of 5X T4 ligase buffer in a reaction) T4 DNA ligase (Invitrogen) 3 µl 9.2) Reclaim the beads against MPC x 1 min and then remove all supernatant (and save it) 9.3) Perform the wash steps (wash reclaim against the MPC x 1 min remove supernatant resuspend in new wash buffer transfer to a new tube) in the following orders --- 300 µl of TWB 300 µl of TWB 200 µl of 1X BB 200 µl of 1X NEBuffer 2 50 µl of 1X NEBuffer 2 9.4) After the last wash, resuspend the beads in ~20 µl (depending on how much DNA you have after AMPure purification step) of 1X NEBuffer 2 and transfer to a new tube 9.5) Set up PCR reactions as follows with different PCR cycle numbers (set up 3-4 different PCR reactions for each sample, for instant 8 cycles, 10 cycles, 12 cycles and 14 cycles) --- purified bead-bound HiC DNA 0.9 1 Rxn 15 PE primer 1.0 (titration) 0.21 3.15 PE primer 2.0 (titration) 0.21 3.15 25mM dntps 0.12 1.8 10X PfuUltra Buffer 1.5 22.5 H2O 11.76 176.4 PfuUltra II Fusion DNA polymerase 0.3 4.5 '--- Stratagene (#600670) total 15 211.5 98ºC 30 se 98ºC 10 sec 65ºC 30 sec X N cycles 72ºC 30 sec 72 C 7min 4 C forever HiC protocol for John Stam Collaboration - Ye Page 11

9.6) Load 7.5µl of each PCR product on 2% gel to check the size range of final library, find out the optimal PCR cycle number and estimate the yield of DNA 9.7) Meanwhile, use PCR products from the highest cycle number as the templates to set up Nhe1 digestion reaction as follows and decide which sample will be proceeded to make final HiC library --- set up control and Nhe1 reactions per sample --- 3 µl DNA per reaction --- incubate at 37 C x 1 hour --- load on 2% to check the quality of the library in term of % of dangling ends 9.8) Perform multiple PCR reactions with sequencing paired end primers using the optimal PCR cycle number (normally generate ~200 300 ng DNA for each library) --- try to increase the DNA input to reduce PCR cycles --- doubling the amount of DNA input should theoretically reduce one PCR cycle --- using 4 times of DNA should theoretically reduce two PCR cycles 9.9) Purify the final PCR products using AMPure XP mixture (use the following procedures if the library size is fine) 9.9.1) Allow AMPure XP mixture to come to room temperature and mix well prior to use 9.9.2) Mix the PCR product with 1.1X volume of AMPure XP thoroughly by pipetting 10 times 9.9.3) Let the mixed samples incubate x 10 min at RT 9.10) Reclaim the beads against the MPC until the solution become clear and then discard the supernatant 9.11) Wash the beads with 500 µl fresh 70% ethanol while the magnetic beads remained against the MPC without disturbing the separated beads 9.12) Repeat step 9.11) once 9.13) Air-dry the beads completely 9.14) Resuspend the beads in ~10-20 µl of EB (I try to keep each concentration around 20 ng/ µl) and incubate at RT x 5 min 9.15) Reclaim the beads against the MPC x 1 min 9.16) Transfer the supernatant containing the purified PCR products to a new tube 9.17) Load 1 ~ 2 µl of each purified PCR product on 2% gel to check the quality of the purified HiC products and the concentration of the HiC library 9.18) Dilute or concentrate the HiC library to the required concentration for sequencing (~ 2-2.5 ng/µl or 10 nm) HiC protocol for John Stam Collaboration - Ye Page 12