alias: GSM1200123
title: GSM1200123: RLPII_phospho5_ab5131.Clone_8.Solexa Input Rep1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: 1. Grow cell cultures in large T-flasks (175-225cm2) to a density of~5x106 cells/ml. Combine the content of 2-3 flasks in one. Immediately proceed with the following steps.   2. Add 37% formaldehyde (Sigma) to final concentration 1.8% directly to the culture incubate for 10min at 250C on a rotating or rocking platform.   3. Stop reaction by adding 1.25M glycine pH 7.0 to each culture to the final concentration of 125mM. Mix well, within next 5 min aliquot cell suspension into 50ml falcon tubes and accumulate them on ice.   4. Pellet fixed cells by spinning for 2? at 1500g, +40C. Use swinging bucket rotor.   5. Resuspend each pellet in 5ml of sterile 1xPBS, pool suspensions together.   6. Pellet the cells by spinning for 2? at 1500g, +40C.   7. Resuspend the cells in 10ml of ChIP wash A solution. Transfer suspension to a new 15ml Falcon tube. Wash fixed cells for 10min at +40C using rotating wheel. Pellet as above.   8. Thoroughly resuspend fixed cells in 10ml of  ChIP wash B solution, wash for 10min at +40C. Use rotating wheel. After treatment with formaldehyde, Drosophila tissue culture cells were lysed with SDS and sonication (on ice) in the presence of protease inhibitors.  The resulting chromatin was treated with non-ionic detergents at physiological concentrations of monovalent cations.  Ribonucleic acid was removed by RNAse A, and the DNA was purified using SDS, proteinase K, and organic extraction, followed by ethanol precipitation. 1. Add 30ml of PAS (50% suspension in RIPA (-PMSF)) to 500ml of crosslinked chromatin. Incubate 1h at +40C.   2. Spin suspensions for 2min at top speed +40C. Transfer supernatants to new tubes. Add 5ml of 100mM PMSF solution in isopropanol to each 500ml aliquot of precleared chromatin.   3. Add appropriate amount of antibody to each reaction. Do not forget to set up no Ab control. Incubate for 15 hours at +40C on rotating weel.   4. Add 40ml of PAS (50% suspension in RIPA (-PMSF)), incubate 3h at +40C on rotating weel.   5. Wash the beads 5 times 10min each with 1ml of RIPA, then one time with 1ml of LiCl ChIP buffer and finally twice with 1ml of TE. To pellet the beads between washes spin samples for 20sec +40C at top speed. Do all the washes at +40C.   6. Resuspend the beads in 100ml TE add 1ml (final 50mg/ml) of RNAse A (10mg/ml) incubate 30min at +370C.   7. Add 7.5ml (final 0.5%) of 10% SDS and 3.8ml (final 0.5mg/ml) of Proteinase K (20mg/ml). Incubate overnight at +370C.   8. Transfer samples at +650C, incubate 6h.   9. Add 4.5ml of 5M NaCl (140mM final). Extract samples with 150ml of phenol-chloroform by vortexing for 30 sec, centrifuge for 10 min at RT, take 120ml of aqueous phase, back-extract organic phase with 150ml of TEN 140 (10mM Tris-HCl pH8.0; 1mM EDTA; 140mM NaCl). Take 150ml of aqueous phase. Combine aqueous phases (you will get 120ml + 150ml=270ml of solution).  10. Extract samples with 300ml of chloroform by vortexing for 30 sec, centrifuge for 5 min at RT. Transfer the upper aqueous phase into the new tube. Add 30ml of 3M NaAc pH 5.0 and 2ml of glycogen (5mg/ml) to aqueous phase. Precipitate DNA with 900ml of EtOH at -700C for 1h.  11. Spin for 10min, top speed at +40C. Wash the pellet in 300ml of 70%EtOH  12. Spin for 10min, top speed at +40C. If you plan to do qPCR analysis only dissolve the pellet in 150ml of pure H2O. If you plan to do both qPCR and microarray hybridization then first dilute DNA pellets in 12ml of pure H2O transfer 4ml of DNA solution to a new eppendorf tube and add 46ml of pure H2O. Use the latter for qPCR and the former for subsequent amplification and labeling. Store DNA solutions at -200C.  13. Prepare dilutions of DNA isolated from the original crosslinked chromatin (also called ?Input DNA? or simply ?Input?) following the chart below. Use the stock with concentration of 0.5% of input DNA per ml of solution (see: ?Isolation of ChIP Input DNA? protocol). Protocol for making libraries for ChIP-seq with Illumina platform, using the TruSeq DNA Sample Prep Kit.http://www.illumina.com/products/truseq_dna_sample_prep_kit.ilmn
identifiers.namespace: GEO
identifiers.id: GSM1200123
attributes.tag: GEO Accession
attributes.value: GSM1200123