Download 1 ChIp protocol

 ChIP protocol (B­cells) ver. 3.3. Bojan Vilagos, I.M.P. Vienna 28thOctober 2008 ChIp protocol (B­cells) (all steps including cells are performed on ice) 1a|
Fill the tubes for organs with PBS1 (3‐5ml) and put them on ice. 2a|
Isolation of lymphocytes. Extract lymph nodes from a mouse2. Figure shows positions of the lymph nodes. 3a|
Press the organs through a cell strainer (using a syringe piston) and wash with few ml of cold PBS (don’t exceed 10ml). *For 1 sample use cells from two mice. Spin down the cells at 1500rpm (300­350 G) for 5‐10min at 4°C. 4a|
Discard the supernatant. (If it remains turbid and full of cells, collect it to new tube and repeat centrifugation.) Resuspend the pellet in 1ml ACK Lysing buffer (lyses the erythrocytes in hypotonic environment) and incubate 2min at RT (or up to 4 min on ice). 5a|
Wash the cells 1x with 5ml PBS (resuspend in 1ml and then fill up) and centrifuge at 1500rpm for 5min at 4°C. After washing resuspend in 1 ml of cold PBS and count the cells3. (Skip step 1 in MACS protocol.) Pause point. Cells in FACS/MACS buffer can be stored on ice. MACS protocol (positive selection) 1|
Resuspend the pellet in 90µl MACS buffer per 107 cells (adjust to 1ml). 2|
Optional. Add Fc‐block antibody (1/500), mix4 and incubate for 5min. 3|
Take two5 aliquots containing approx. 50‐100k cells which will be later used for FACS analysis. 4|
Proceed by one option: a. Add biotinilated antibody, incubate on ice for 15‐30min and wash with MACS buffer. Afterwards add streptavidin coupled magnetic beads, incubate for 15‐30min and wash with MACS buffer. b. Add antibody coupled with magnetic beads, incubate for 15‐30min and wash with MACS buffer. c. Use lineage specific Ab cocktail* (find data at the end of protocol) and incubate for 30‐45min in dark (mix occasionally). Wash cells 1x with 10ml of buffer and centrifuge at 1500rpm for 5min at 4°C. Aspirate supernatant and resuspend the cells in 1 ml of MACS buffer. Add Anti‐PE‐MicroBeads and incubate 20‐30min. *Important note: Each antibody and beads have different specifications (e.g. 20µl Anti­PE­
MicroBeads should be added per 107 cells)! 1 Use MACS or FACS buffer instead to keep cells more viable. 2 Up to 50 million cells can be extracted from 1 (uninfected) mouse by taking most of lymph nodes. 3 Use 2µl of sample in 10ml of counting fluid. (That makes 5000 fold dilution; dead cells and erythrocytes 0­
5µm, lymphocytes 5­7.5µm, lymphoblasts 7.5­10µm in diameter). 4 Only when working with mature B cells. 5 pre‐sortstained and pre‐sortunstained. 1 2 ChIP protocol (B­cells) ver. 3.3. Bojan Vilagos, I.M.P. Vienna 28thOctober 2008 5|
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Wash cells 1‐3x by adding 1‐2ml of buffer per 107 cells (10 ml) and centrifuge at 1500rpm for 5min at 4°C. Aspirate supernatant completely. Resuspend up to 108 cells in 500µl of buffer (3ml). *Important note: Before resuspending the cells take in mind capacity of column (e.g. LS columns when used for positive selection: max. 2x109 cells and max. 108 labelled cells)! Place the column6 on magnetic holder rinse it with 5ml of buffer. Place the tube for flow through collection. Optional. Place pre‐selection filter on the column (removes clumps). Apply cell suspension onto the column. Collect cells that pass through. Wash column 3x with 3ml of MACS buffer, also collect this fraction. (Take aliquot7 for FACS analysis and count cells.). In case of positive selection. Remove column from separator and place it on a suitable collection tube. Add 5ml of MACS buffer and immediately flush out magnetically labelled cells by firmly pushing the piston. (Count cells and take aliquot for FACS analysis.) 7|
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Cross­linking If MACS or FACS buffers were used (they contain proteins that would interfere with cross­
linking) wash cells and later resuspend them using cold PBS. 13|
Cross‐link proteins and DNA by adding 1% formaldehyde to the solution and incubate 10min at RT (27µl of 37% formaldehyde per 1ml of solution). 14|
Quench formaldehyde by adding 125mM glycine for 5min at RT (141µl of 1M glycine per 1ml of solution). 15|
Wash the cells 1‐2x with 5‐10ml of PBS (resuspend in 1ml and then fill up) and centrifuge at 1500rpm for 5min at 4°C. Lysis 16|
Resuspend the pellet in 2ml IP Lysis buffer8 containing protease and/or phosphatise inhibitors if needed and incubate for 30min at 4°C. Sonication (in Falcon® 15ml tubes) 17|
To shear chromatin, sonicate in 2ml IP Lysis buffer per 20‐50 million cells (one IP sample). *Sonicate in ice‐water (length9 and number of pulses and relaxation periods needs to be established for each cell line), max. power. 18|
Centrifuge the lysates at 13000rpm for 10min at 4°C and collect the supernatant containing sheared DNA to a new microcentifuge tube. Pause point. Lysates can be stored in fridge. 6 LS column use in this protocol. 7 This is the unlabeled cell fraction (flow‐throughstained). 8 2ml per sample (containing ≈20 million cells) it is possible to lyse in 1 tube and divide samples later. 9 For B‐cells use 22x 30sec long pulses and 30sec long relaxation periods. ChIP protocol (B­cells) ver. 3.3. Bojan Vilagos, I.M.P. Vienna 28thOctober 2008 Shearing Check Option 1 (steps 1b­7b) 1b| Take 50‐100µl (equivalent to 0.5‐1 million cells) of sheared chromatin aliquot and add H2O to 400µl. This will be used to determine shearing efficiency. 2b| Add 5µl of 10µg/µl RNase A and incubate for 30‐60min at 37°C. 3b| Add 5µl of 20µg/µl proteinase K and shake for 30‐60min at 55°C (shake, 750rpm). 4b| Reverse cross‐links at 65°C for 6h (or overnight). 5b| Recover DNA by performing phenol : chloroform : isoamyl‐alcohol (25:24:1) extraction. Add 1/1 volume of organic solvent to your sample, vortex, centrifuge with 13000rpm for 10min and collect water phase afterwards. 6b| Precipitate DNA by adding 2 – 2.5 volumes of 99% ethanol, 1/10 volume 3M NaAc, 1µl 20µg/µl glycogen (to better visualise DNA pellet) and incubating for 2h at ‐20°C. Afterwards centrifuge at 13000rpm for 20min at 4°C and discard the supernatant. 7b| Wash the DNA pellet with 1ml of 70% ethanol, centrifuge with 13000rpm for 3min and air dry it (37°C 10min). Resuspend in 50µl of TE buffer. Option 2 (steps 8b­14b) 8b| Take up to 200µl of sheared chromatin aliquot and add H2O to 400µl. Add 0.05g of Chelex 100 slurry and boil (95°C) for 10 min (mix sample). For high chromatin concentrations (aliquot less than 100µl) use 10% Chelex 100 slurry directly to fill up to 500µl. 9b| Optional. Proteinase K treatment. Wait for samples to cool after boiling and add 5µl of 20µg/µl proteinase K to each sample. Shake for 30min at 55°C (1000rpm). Boil (95°C) again for 10min and cool afterwards. 10b| Centrifuge condensate at 13000rpm for 1min at 4°C. Transfer supernatant (400µl) to a new tube. 11b| Optional. Add 120µl of water to beads, vortex and centrifuge contents down at 13000rpm for 1min at 4°C. Collect supernatant (120µl) and pool with the previous supernatant. 12b| Precipitate DNA by adding 2 – 2.5 volumes of 99% ethanol, 1/10 volume 3M NaAc, 1µl 20µg/µl glycogen (to better visualise DNA pellet) and incubating for 2h at ‐20°C. Afterwards centrifuge at 13000rpm for 20min at 4°C and discard the supernatant. 13b| Wash the DNA pellet with 1ml of 70% ethanol, centrifuge with 13000rpm for 3min and air dry it (37°C 10min). Resuspend in 50µl of TE buffer. 14b| Use 20µl for electrophoresis in 2% agarose gel. Shearing is good if majority of DNA is below 500bp and smear has a peak between 200 and 300bp. Immunoprecipitation Important note. Add protease inhibitors10 to all buffers used before ChIP Elution buffer and work in the cold room until elution of immune complexes. 19|
Optional. Quantify chromatin at 260nm. Use 200µg to 400µg of chromatin per IP sample. 20|
Take 1/100 of lysate for input and dilute the rest 10 fold using ChIP Dilution buffer. 21|
Pre‐clear chromatin solution by adding 50µl protein A coated beads, rotate for 2h. 22|
Centrifuge the solution with 1500rpm for 5min and harvest supernatant. 10 Add protease inhibitors shortly before use of a buffer because they are functional only for 30min, at +4°C. 3 4 ChIP protocol (B­cells) ver. 3.3. Bojan Vilagos, I.M.P. Vienna 28thOctober 2008 23|
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Divide chromatin solution in 2 equal samples. Immunoprecipitate one sample with 5µg of an antibody*, rotating overnight. Treat sample without antibody in same way as IP sample. Harvest immune complexes with 50µl protein A coated beads, rotate for 2h. Wash beads by adding 1ml of a buffer and rotate for 5‐10min. Afterwards spin down at 2000rpm for 2min (micro­centrifuge) and discard supernatant. Washing order is: 1x ChIP Low Salt buffer 1x ChIP High Salt buffer 1x ChIP LiCl buffer 2x ChIP TE buffer Elute immune complexes by adding 250µl ChIP Elution buffer (prepared fresh), shake 15min at RT. Spin down the beads at 2000rpm for 2min and transfer supernatant to a new microcentrifuge tube. Repeat elution step and combine eluates. Add ChIP Elution buffer up to 500µl to input sample and treat it from now on as IP sample. Add 16µl 5M NaCl and 12.5µl 10µg/µl RNase A, incubate 1h at 37°C and afterwards reverse cross‐links at 65°C for 6h (or overnight). Add 16µl 1M Tris pH 6.5, 8µl 0.5M EDTA, 12.5µl 20µg/µl Proteinase K and incubate 1h at 45°C. Recover DNA by performing 1x phenol : chloroform : isoamyl‐alcohol (25:24:1) extraction and 1x chloroform extraction. Add 1/1 volume of organic solvent to your sample, vortex, centrifuge with 13000rpm for 10min and collect water phase afterwards. Precipitate DNA by adding 2 – 2.5 volumes of 99% ethanol, 1/10 volume 3M Na‐Acetate, 1µl 20µg/µl glycogen (to better visualise DNA pellet) and incubating for 4h (or overnight) at ‐20°C. Afterwards centrifuge at 13000rpm for 20min at 4°C and discard the supernatant. Wash the DNA pellet with 1ml of 70% ethanol, centrifuge with 13000rpm for 3min and air dry it (37°C 10min). Resuspend in 50µl of TE buffer. Perform directed ChIP PCR analysis of target genes* as shown on figure. Quantify chromatin using PicoGreen® Assay. There should be at least 10ng of chromatin in 30µl sample for one Solexa® sequencing run. ChIP protocol (B­cells) ver. 3.3. Bojan Vilagos, I.M.P. Vienna 28thOctober 2008 MACS depletion Ab cocktail for mature B­cells Optimized for 80­90million of total lymphocytes extracted from lymph nodes (two mice). Staining volume 1ml. Volume/µl 8 5 6 1 2 2 4 4 1 Type of antibody CD3ε‐PE CD4‐PE CD8α‐PE CD11b‐PE (Mac1) CD11c‐PE Gr1‐PE TCRβ‐PE CD5‐PE NK1.1‐PE Dilution for FACS commercial home made home made 1/500 1/2000 1/1000 commercial 1/10000 commercial home made home made home made commercial 1/300 1/500 1/500 1/500 1/200 *Important note. Up to 50% of initial cells will be lost during the preparation and washings before MACS, and after sorting 1/3 of sample will remain as mature B­cells. Arround 20 million cells are needed for one ChIP sample11 . From one uninfected 6­8 weeks old mouse (C57BL/6J) around 4­7 million mature B­cells can be extracted using this protocol. To reduce los of cells use minimal number of washing steps and don’t put organs from more than two mice in one tube. 11 There isn't a consensus about the ammount of chromatin needed for succesfull sequencing and it also depends whether it is a chromatin mark or transcription factor pull‐down. 5 6 ChIP protocol (B­cells) ver. 3.3. Bojan Vilagos, I.M.P. Vienna 28thOctober 2008 Buffers for ChIP final concentration: for 100ml use: Lyses Buffer 1% SDS 10mM EDTA 50mM Tris‐HCl pH 8.1 10ml 10% SDS 2ml 0.5M EDTA 5ml 1M Tris‐HCL pH8.1 0.01% SDS 1.1% Triton X‐100 1.2mM EDTA 16.7mM Tris‐HCl pH 8.1 167mM NaCl 100µl 10% SDS 11ml 10% Triton X‐100 0.24ml 0.5M EDTA 1.67ml 1M Tris‐HCl pH 8.1 3.34ml 5M NaCl 0.1% SDS 1% Triton X‐100 2mM EDTA 20mM Tris‐HCl pH 8.1 150mM NaCl 1ml 10ml 0.4ml 2ml 3ml 10% SDS 10% Triton X‐100 0.5M EDTA 1M Tris‐HCl pH 8.1 5M NaCl 0.1% SDS 1% Triton X‐100 2mM EDTA 20mM Tris‐HCl pH 8.1 500mM NaCl 1ml 10ml 0.4ml 2ml 10ml 10% SDS 10% Triton X‐100 0.5M EDTA 1M Tris‐HCl pH 8.1 5M NaCl 0.25M LiCl 1% NP‐40 1% deoxcholate 1mM EDTA 10mM Tris‐HCl pH 8.1 5ml 10ml 1g 0.2ml 1ml 5M LiCl 10% NP‐40 deoxycholic acid12 0.5M EDTA 1M Tris‐HCl pH 8.1 ChIP Dilution buffer Low Salt Immune Complex High Salt Immune Complex LiCl Immune Complex Elution Buffer 1% SDS 100mM NaHCO3 Protein A beads Resuspend 0.04g of protein A coated beads in 200µl H2O and wash them 3x using 1xTE buffer. Afterwards resuspend the beads in solution consisting of 4µl Salmon Sperm DNA (10mg/ml), 20µl BSA (10mg/ml), 10µl NaAzid (2%) and 174µl 1xTE. 12 disolves weakly