title: GSM3137715: Midbrain neuroepith. H3K9me3 ChIP Rep 1; Mus musculus; ChIP-Seq
library_construction_protocol: B6C3F1 female were superovulated by intraperitoneal injection (5 IU pregnant mare serum – PMSG, Prospec, cat# hor-272) followed, 48h later, by a second injection (5 IU human chorionic gonadotropin - HCG, Peprotech cat#: 100-39-10UG) and then bred to C3H or Pdx1-eGFP or Ins-RFP males. To generate single cells for FACS, embryos at different developmental stages (i.e. e8.25, e10.5, e11.5, e18.5) were dissected out of decidua and collected in 1.5ml tubes containing 1X PBS on ice. For e10.5 and e11.5 embryos, most of the dorsal part (somites) was removed. E18.5 pancreata, clearly visible and easy to distinguish, were dissociated from the rest of the organs. Collected samples were dissociated with 0.05% Trypsin-EDTA (Life Technologies, #25300-054) for 5 min at 37C while gently shaking. The reaction was stopped by adding 0.4 volumes of FBS (Hyclone, SH30071). Explants were homogenized by gentle pipetting. Cells were spun down at 1500rpm for 5min at 4C, and the pellet was resuspended in 320 ul of IMDM (Life Technologies, #12440-053) (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). Twenty (20) ul were transferred into a new tube and 280 ul IMDM were added to it (negative control tube - i.e. samples non incubated with the primary antibody). E8.25 definitive endodermal cells were purified using an antibody detecting ENDM1 (dilution: 1:50) surface antigen (31–33). E10.5 hepatoblasts were sorted from Pdx1-GFP transgenic embryos, staining the cells for the surface marker Liv2 (MBL, cat# D118-3, dilution: 1:100). At the same stage pancreatic precursor Pdx1-GFP positive cells were collected. A subpopulation of Liv2+/Pdx1-GFP+ cells was detected at E10.5, as previously reported (33). These cells were not considered for further analysis. E11.5 hepatoblasts were sorted from wt embryos using the same antibody and conditions as for e10.5 embryos. Cells were incubated for 30-40 min with primary antibody. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 300 ul of IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122). PE-conjugated goat anti rat IgG (Biolegend 405406) was added (dilution 1:300). Samples were mixed well and incubated for 30 min on ice in the dark. Tubes were flicked every 5-8min. One (1) ml IMDM (supplemented with L-glut – Life Technologies #25030-081, 0.1%BSA - Sigma A9418-50G, 2.5% FBS - Hyclone, SH30071, 1X pen/strep – Life Technologies, #15140-122) was added to both the sample and negative ctrl tube. Cells were spun down at 1500rpm for 5min at 4C and the pellet was resuspended in 400-800ul 1XPBS + 3% BSA (Sigma A9418-50G). Samples were filtered through a cell strainer (FALCON 5ml polystyrene round-bottom tube with cell-strainer cap, #352235). Samples were kept on ice until sorting. Mature beta cells were isolated from Ins-RFP transgenic embryos as described (34).Two (2) -month old mice were anesthetized using isoflurane (Butler Animal Health Supply, #029405) and monitored by paw pinch.  A V-shaped incision was used to reveal the abdominal cavity, the intestines moved aside, and a 22 or 24 gauge catheter (Midwest Veterinary Supply, #381423) was used to cannulate the venae cavae while liver perfusion media pumps into the liver, and the portal vein is immediately severed. 37°C liver perfusion media (Invitrogen #17701-038) and then liver digest media (Invitrogen #17703-034) are perfused through the liver (45mL each per animal).  Livers are dissociated with a cutting motion with cell scrapers in William's E Medium (Sigma W4128) and strained through a 100μm filter (BD 352360).   Hepatocytes are loosely pelleted by centrifugation at 50g for 5 min at 4°C. Twenty-five ul of the hepatocytes pellet were transferred into RLT buffer (RNeasy minikit). One (1) month old control and triple knockout (TKO) mutant liver were dissected and cut in pieces. A small piece was transferred to 1.5 ml tube. Samples were minced using dissecting scissors. One (1) ml ACK (Life Technologies, #A10492-01) solution was added and samples were incubated 5-7 min on ice. Samples were spun down at 1,500 rpm for 5 min at 4C; pellet was resuspended in 500 ul 1XPBS and dounced with microcentrifuge tubes pellet pestle (DWK Life Sciences – Kimble - #7495150000) for 10-15 sec on ice. Samples were spun down at 1,500 rpm for 5 min at 4C.Pellet was resuspended in RLT buffer (RNeasy minikit).31.P. Gadue et al., Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm. Stem Cells Dayt. Ohio. 27, 2103–2113 (2009).32.C.-R. Xu et al., Chromatin “prepattern” and histone modifiers in a fate choice for liver and pancreas. Science. 332, 963–966 (2011).33.C.-R. Xu et al., Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification. EMBO J. 33, 2157–2170 (2014).34.D.-S. Li, Y.-H. Yuan, H.-J. Tu, Q.-L. Liang, L.-J. Dai, A protocol for islet isolation from mouse pancreas. Nat. Protoc. 4, 1649–1652 (2009). We provide here a detailed protocol describing how to prepare low cell number sample for chromatin-related experiments.  A) Notes:  The following protocol is modified from O'Geen, et al. (37) and recommended for small cell number ChIP-seq and is based on successful 3 x 10^4 cells for ChIP-seq with H3K9me3 and H3K27me3 antibodies in mouse sorted cells isolated by FACS with cell surface markers at different developmental stages (i.e. e8.25, e10.5). Please note for optimal DNA recovery when purifying ChIP, use a Phenol Chloroform method. This protocol was chosen due to its high specificity and low background.     B) Buffers:  Cell Lysis Buffer (make 10 ml fresh each time)  3mM MgCl2  10mM NaCl  10mM Tris, pH 7.4  0.1% Igepal (Sigma Aldrich, cat #I8896)  Add protease inhibitors (1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001-, per 10 ml Buffer).     Nuclei Lysis Buffer (prepare it fresh each time)   50mM Tris-Cl pH 8.0  10mM EDTA  1% SDS (Lonza, #51213)  Before use, add protease inhibitors (1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001- per 10 ml Buffer. Never store at 4C).    IP Dilution Buffer (RIPA)   50mM Tris pH 7.4  150mM NaCl  1% Igepal (Sigma Aldrich, cat #I8896)  0.25% Sodium Deoxycholate (Sigma Aldrich, cat #D6750)  1mM EDTA  Add protease inhibitors (1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001- per 10 ml Buffer).    IP Wash Buffer 1 (RIPA without protease inhibitors; place it on ice after preparation)  50mM Tris pH 7.4  150mM NaCl  1% Igepal (Sigma Aldrich, cat #I8896)  0.25% Deoxycholic Acid (Sigma Aldrich, cat #D6750)  1mM EDTA    IP Wash Buffer 2 (place it on ice after preparation)  100mM Tris-Cl pH 9.0  500mM LiCl  1% Igepal (Sigma Aldrich, cat #I8896)  1% Deoxycholic acid (Sigma Aldrich, cat #D6750)    IP Wash Buffer 3 (IP Wash Buffer 2 plus NaCl; place it on ice after preparation)  100mM Tris-Cl pH 9.0  500mM LiCl  1% Igepal (Sigma Aldrich, cat #I8896)  1% Deoxycholic acid (Sigma Aldrich, cat #D6750)  150mM NaCl    Elution Buffer   50mM NaHCO3  1% SDS (Lonza, #51213)    C) Cell crosslinking:  The following has been optimized for crosslinking sorted cells:  1. Use FACs to sort > 104 cells into 1.5 ml tube, containing 50 ul 1X PBS supplemented with 1% BSA (Sigma A9418-50G) and 20% FBS  (Hyclone, SH30071). Spin down the cells at 1700rpm for 12-15 min at 4C. Resuspend the pellet in 200-300ul PBS freshly prepared. Prepare 3 x 104 cells aliquots in 500ul PBS or alternatively, if you have a lot of cells, prepare 1-3 x 105 cells aliquots.  2. Add 13.5 ul of 37% formaldehyde (Fisher Chemical F79-500) per 500 ul sample. Incubate exactly 10 min at room temp on rotating platform.  3. Stop crosslinking reaction by adding 36 ul of 2M glycine (Fisher BioReagents BP381-1) to the sample. Incubate 5 min at room temp on rotating platform.  4. Centrifuge at 2000 RPM for 15 min at 4C  5. Wash cells twice in cold PBS supplemented with 2% FBS  (Hyclone, SH30071). For each wash, centrifuge at 2000 RPM for 5 min. Slowly aspirate supernatant with pipetman, leaving approximately 10 ul of solution behind. Be careful to not disturb the pellet.       D) Preparation of chromatin from crosslinked cells:  1. If you have > 105 cells, resuspend cells in 300 ul freshly prepared cell lysis buffer and incubate on ice for 15 min. Centrifuge at 5000 RPM for 5 min at 4 C. Repeat centrifugation if cell lysate is not visible. Remove supernatant with pipetman, leaving pellet. Leave approximately 10 ul of supernatant. If starting with < 105 cells, proceed to step 2.  2. Prepare aliquots of 3 x 104 cells in 130 ul Nuclei Lysis Buffer.  3. Incubate on ice for 15 minutes, flash freeze (use dry ice or liquid nitrogen), then thaw to room temperature to aid in lysis. (You may keep chromatin at -80 C until a future day and then thaw to room temperature).  4. Sonicate with predetermined conditions so that chromatin is between 200 and 500 bp in length. If using Covaris (S220 Focused Ultrasonicator) for chromatin sonication apply the following parameters:  Incident peak power: 200  Duty Cycle: 5%  Burst/Cycle: 200  Time: 10-12min  5. Centrifuge sonicated chromatin for 10 min at 14K RPM at 4C, and collect supernatant and either use immediately in ChIP experiment or freeze at -80C.    E) Checking sonicated chromatin:  1. Collect about 3*103 cells and resuspend them in elution buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS - Lonza, #51213)  2. Reverse crosslink at 65 C o/n on heat block  3. Add 200 ul TE buffer (to dilute SDS - Lonza, #51213)  4. Add 8 ul of 10 mg/ml RNase A (Roche, #10109169001) and incubate samples for 1.5-2 h at 37 C  5. Add 4ul Proteinase K (20mg/ml stock) (Roche, #03115828001) and incubate 1.5-2 h at 55C  6. Precipitate DNA with standard phenol-chloroform protocol  7. Transfer the aqueous phase to a new tube  8. Add 16 ul of 5 M NaCl, 1.5 ul of 20 mg/ml glycogen (Roche, #10901393001) and 800 ul 100% EtOH  9. Place the samples at -80 C for a couple of hours (you can do this step o/n)  10. Pellet DNA spinning the samples at 15,000 rpm for 12 min at 4 C (pellet is faint!)  11. Wash with 500 ul 80% EtOH, once  12. Pellet DNA spinning the samples at 15,000 rpm for 12 min at 4 C  13. Air dry the pellet and resuspend in 15 ul TE buffer (remove EtOH)  14. Spin vacuum the samples and resuspend them in 3ul H2O containing 10% glycerol  15. Prepare 1% agarose gel (as thin as possible) without Ethidium Bromide  16. Load the samples and marker   17. Run the gel at 90V for 40-45 min  18. Stain the gel (10 ug/ml final concentration) for 15 min RT while shaking  19. Wash the gel 5 min RT while shaking in ddH2O  20. Image the gel.  Note: the sonication can be checked with the Bioanalyzer (Agilent Technologies G2939AA) by resuspending the samples from step 14 in water (3 ul).    Chromatin Immunoprecipitation (ChIP) on low cell number samples:  We provide here a detailed protocol describing how to perform chromatin immunoprecipitation and how to obtain samples ready to be used for libraries preparation starting from low cell number samples.    A) Immunoprecipitation  1. Transfer sonicated chromatin into 0.6 ml Axygen Maxymum Recovery microtubes (MCT-060-L-C, #311-03-051). Dilute chromatin with Dilution Buffer to a final volume of 500 ul. For input, take 5 ul (1%) from the 500ul containing sheared chromatin and transfer to new tube containing 100 ul elution buffer.  2. Add 1 ug of primary ChIP-grade antibody and incubate overnight on a rotating platform at 4 deg.  3. Add 15 ul Cell Signaling ChIP-grade Protein G magnetic Beads (Invitrogen #10004D) and incubate 2 hours on rotating platform at 4 deg.  4. Transfer chromatin and beads to a pre-chilled 1.5 ml microtubes and allow beads to separate in magnetic rack (Thermo Fisher Scientific, #12321D) for > 30 seconds.  5. Remove supernatant with pipetman. Using suction may cause you to lose beads and sample.   6. Remove tube from rack, resuspend beads in IP Wash Buffer 1, and wash by pipetting up and down 12 times.  7. Place tubes in magnetic rack  (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.  8. Repeat steps 5-7 once.  9. Remove supernatant with pipetman.  10. Remove tubes from rack, resuspend beads in IP Wash Buffer 2, and wash by pipetting up and down 12 times.  11.  Place tubes in magnetic rack (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.  12. Repeat steps 9-11 2 times.  13.  Remove supernatant with pipetman.  14.  Remove tubes from rack, resuspend beads in IP Wash Buffer 3, and wash by pipetting up and down 12 times.  15.  Transfer beads and chromatin to a new 1.5 ml microtube.  16.  Place in magnetic rack (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.  17.  Remove tubes from rack and resuspend in 100 ul Elution Buffer.  18.  Shake at RT at a setting of 1300 RPM for 1 hour.  19.  Place tubes in magnetic rack (Thermo Fisher Scientific, #12321D) and allow beads to separate for > 30 seconds.   20.  Collect supernatant and add 12 ul 5M NaCl.  21. Thaw the input and add 12ul 5M NaCl  22.  Incubate 4 hours to overnight at 65 deg.  23.  Add 3 ul of 10 mg/ml RNase A (Roche, #10109169001) to each sample and incubate at 37 C for 30 min.    B) Phenol Chloroform sample purification  1. Spin down Phase Lock Gel Light 1.5 ml tube (5 Prime, QuantaBio, #2302820) at 14000 rpm for 30sec.  2. Add 100ul TE buffer to each samples (including input) and mix gently (total volume 200ul)  3. Mix 200 ul ChIP product and 200 ul Phenol/Chloroform/Isoamyl Alcohol by pipetting about 10 times and add to Phase Lock Gel Light 1.5 ml tube. Do not vortex.  4. Centrifuge at room temperature for 5 minutes at 14000 RPM.  5. Optional: Add an additional 200 ul of Phenol/Chloroform/Isoamyl Alcohol and mix by pipetting.  6. Centrifuge at room temperature for 5 minutes at 14000 RPM.  7. Collect aqueous solution on top and add to a new tube (should be about 200 ul). If the volume is less than 200 ul, add Buffer TE to reach 200 ul.   8. Add 8 ul 5M NaCl, 10 ug glycogen, and 400 ul cold 100% ethanol, and mix.  9. Precipitate DNA in ethanol at -80 overnight at least for best recovery.   10. Centrifuge at 4 C for 10 min at 11000 RPM.  11. Aspirate supernatant carefully, without disturbing the very small white pellet. To be safe, you may leave 5 ul at the bottom.  12.  Wash with 500 ul 80% ethanol by pipetting five times.  13.  Centrifuge at 4 C for 10 min at 11000 RPM.  14.  Aspirate supernatant carefully, without disturbing the very small white pellet.   15.  Air dry 5 minutes, or until ethanol has evaporated, but pellet isn't completely dry. If pellet is too dry, it will be difficult to resuspend.  16. Resuspend pellet in 30-40 ul Qiagen EB. It is now ready for qPCR. For library preparation resuspend the pellet in less volume (e.g. 10ul). Libraries were generated using ThruPLEX DNA-seq 48S Kit (Rubicon Genomics, #R400427). Libraries were quantified by qPCR using the NEBNext Library quantification kit for Illumina (NEB, #E7630L). Libraries were diluted to 8 nM and pooled for multiplexing. The diluted concentrations and were quantified a second time using NEBNext kit. Libraries were denatured in 0.2 M NaOH and loaded into NextSeq 75-cycles High Output v2 Kit (Illumina, FC-404-2005) cartridge at a concentration of 2.5-3 pM. Sequencing runs were performed in an Illumina NextSeq 500 machine
attributes.tag: GEO Accession
attributes.value: GSM3137715