Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Three alternative chromatin immunoprecipitation (ChIP) protocols were successfully tested in combination with ChIPmentation. These protocols use different fixation, sonication, lysis, and washing conditions, making it possible to use ChIPmentation with essentially any ChIP-grade antibody; ChIP version 1 (H3K4me3, H3K27me3): Cells were washed once with PBS and fixed with 1 % paraformaldehyde in up to 1 ml PBS for 5 minutes at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 x g for 10 minutes at 4 °C and washed twice with up to 1 ml ice-cold PBS supplemented with 1 μM PMSF. The pellet was lysed in Cell Lysis Buffer (50mM HEPES/KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 % Glycerol, 0.5 % NP-40, 0.25 % Triton X-100, 1x protease inhibitors (Sigma)) for 10 minutes on ice. Nuclei were isolated by spinning the lysed cells for 10 minutes at 1000 x g at 4 °C, the supernatant was discarded and the pellet was resuspended in Sonication Buffer (10 mM Tris-HCl pH 7.6, 1mM EDTA, 0.1 % SDS) and sonicated in a 130 μl microTUBE (for up to 3 x 106 cells) on a Covaris S220 for 12 minutes until most of the fragments were 200-700 base pairs long (settings: duty cycle 2 %, peak incident power 105 Watts, cycles per burst 200). Lysates were centrifuged at full speed for 5 minutes at 4 °C and the supernatant was transferred to a new tube. The lysate was diluted to 200 μl per IP to a buffer composition of 20 mM HEPES, 0.1 % SDS, 1 %Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and incubated with an antibody against H3K4me3 (1 μg/IP, Diagenode C15410003-50) or H3K27me3 (1 μg/IP, Millipore 07-449) over night at 4 °C on a rotator. 20 μl of Protein A Magnetic Beads were blocked over night with 0.1 % BSA in PBS and added to the IP the next day for 2 hours on a rotator at 4 °C to capture the immunoprecipitated fragments. The immunoprecipitated chromatin was washed subsequently with WBI (20mM HEPES, 150mM NaCl, 0.1 % SDS, 0.1 % DOC, 1 % Triton X-100, 1 mM EDTA, 0.5mM EGTA) (twice), WBII (20 mM HEPES, 500 mM NaCl, 0.1 % SDS, 0.1 % DOC, 1 % Triton X-100, 1 mM EDTA, 0.5 mM EGTA) (once), WBIII (20 mM HEPES, 250 mM LiCl, 0.5 % DOC, 0.5 % NP-40, 1 mM EDTA, 0.5 mM EGTA) (once) and WBIV (20 mM HEPES, 1 mM EDTA, 0.5 mM EGTA) (twice). Then beads were incubated with 70 μl elution buffer (0.5 % SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 8.0) containing 2 μl of Proteinase K (NEB). Beads in Elution Buffer were incubated for 1 hour at 55 °C and 8 hours at 65 °C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 hour at 55 °C. Finally, DNA was purified with SPRI AMPure XP beads (ratio sample:beads 1:2) or Qiagen MinElute columns; ChIP version 2 (H3K4me1, and H3K36me3 and REST): Cells were washed once with PBS and fixed with 1 % paraformaldehyde in up to 1.5 ml PBS for 10 minutes at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 x g for 10 minutes at 4 °C and washed twice with up to 1 ml μl ice-cold PBS supplemented with 1 μM PMSF. The pellet was lysed in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 140 mM NaCl, 1 % Triton x-100, 0.1 % SDS, 0.1 % DOC, 1x protease inhibitors (Sigma)) and sonicated in a 1 ml milliTUBE in a Covaris S220 for 30 minutes until most of the fragments were 200-700 base pairs long (settings: duty cycle 5 %, peak incident power 140 Watts, cycles per burst 200). Lysates were centrifuged at full speed for 5 minutes at 4 °C. In the meantime, 50 µl beads (10 µl for low-input ChIPmentation) were blocked and conjugated to an antibody by washing and resupsending them 2 times in PBS/0.5 % BSA/0.5 % Tween-20). A specific antibody was added and bound to the beads by rotating > 1 h at room temperature. Antibodies used in this study were H3K4me1 (1 µg/IP, Diagenode pAb-194-050), H3K36me3 (1 µg/IP, Diagenode pAb-192-050) and REST (10 µg/IP, Millipore 07-579). The supernatant was transferred to a 0.5 PCR tube and per ChIP 50 μl of blocked antibody conjugated magnetic protein A beads were added and incubated for 3 hours at 4 °C. Immunoprecipitation beads were washed subsequently with 150 μl RIPA (twice), RIPA-500 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 500 mM NaCl, 1 % Triton x-100, 0.1 % SDS, 0.1 % DOC,) (twice), RIPA-LiCl (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 1 % Triton x-100, 0.5 % DOC, 0.5 % NP40) and TE pH 8.0 (twice). Then beads were then incubated with 70 μl elution buffer (0.5 % SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 8.0) containing 2 μl of Proteinase K (NEB). Beads in Elution Buffer were incubated for 1 hour at 55 °C and 8 hours at 65 °C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Finally, DNA was purified with SPRI AMPure XP beads (ratio sample:beads 1:2) or Qiagen MinElute columns; ChIP version 3 (H3K27ac, PU.1, CTCF and GATA1): Cells were washed once with PBS and fixed with 1% paraformaldehyde in up to 1.5 ml PBS for 5-10 minutes at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 x g for 10 minutes at 4 °C and washed twice with up to 1 ml μl ice-cold PBS supplemented with 1 μM PMSF. The pellet was lysed in buffer L3B (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1 % Na-Deoxycholate, 0.5 % N-lauroylsarcosine, 1 x protease inhibitors (Sigma)) and sonicated in a 1ml milliTUBE in a Covaris S220 for 20 minutes until most of the fragments were 200-700 base pairs long (settings: duty cycle 5 %, peak incident power 140 Watts, cycles per burst 200). Lysates were supplemented with 1 % Triton-X-100 and centrifuged at full speed for 5 minutes at 4 °C. In the meantime, beads were blocked and conjugated to an antibody by washing them 2 times in PBS/0.5 % BSA and resuspending 50 μl of beads per IP (10 µl beads for low-input ChIPmentation) in 200 μl of PBS/0.5 % BSA. A specific antibody was added and bound to the beads by rotating >1h at room temperature. Antibodies used in this study were H3K27ac (2 µg, Diagenode pAb-196-050) PU.1 (5 μg/IP, Santa Cruz sc-352), CTCF (10 μl/IP, Millipore 07-729) and GATA1 (4 µg/IP and 2µg for low-input, Abcam ab11852). The supernatant was transferred to a 0.5 PCR tube and per ChIP 50 μl of blocked antibody conjugated magnetic protein A beads were added and incubated for 3 hours at 4 °C. Immunoprecipitation beads were washed subsequently with 150 μl TF-WBI (20 mM Tris-HCl/pH 7.4, 150 mM NaCl, 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA) (twice), TF-WBIII (250 mM LiCl, 1 % Triton X-100, 0.7 % DOC, 10 mM Tris-HCl, 1 mM EDTA) (twice) and TET (0.2 % Tween-20, 10 mM Tris-HCl/pH 8.0, 1 mM EDTA) (twice). Then beads were incubated with 70 μl elution buffer (0.5 % SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2 μl of Proteinase K (NEB). Beads in Elution Buffer were incubated for 1 hour at 55 °C and 8 hours at 65 °C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 hour at 55 °C. Finally, DNA was purified with SPRI AMPure XP beads (ratio sample:beads 1:2) or Qiagen MinElute columns. Standard ChIP-seq library preparation: Purified ChIP DNA was end-repaired using the NEBNext End Repair Module (NEB) according to manufacturer’s instruction. Clean-up was done using Ampure XP beads (Agencourt) according to manufacturer’s instruction. Fragments were A-tailed using Klenow (3′→ 5′ exo-) polymerase (Enzymatics), and TruSeq-compatible adapters were ligated using T4 DNA Ligase (Enzymatics). The final library was size-selected using Ampure XP beads to remove adapter dimers; ChIPmentation library preparation: ChIPmentation is c