GSM6703432: AGGAA, rep1; Escherichia coli; ChIP-Seq
To enable immune-precipitation, a C-terminal 3x FLAG tag was added to dCas9 and the gene cloned under the control of a ptet promoter on plasmid pLC133. Samples were prepared from E. coli MG1655 harboring pLC133 and psgRNAcos plasmids with 3 different guide RNAs, a control guide targeting lacZ, the AGGAA_non1 guide or the ACCCA_non1 guide. Cells were grown at 30 degree and induced with aTc when OD600 reaches 0.2. When OD600 reaches 1.0, formaldehyde (final concentration: 1%) was added to fix the cells. After that, glycine was added to block formaldehyde. Samples were further washed with PBS and pelleted.   Samples were then resuspended in 1 ml of IP lysis buffer (50 mM Hepes pH 7.5, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Triton-X100, 10% Glycerol), 20 μl 0.1 M phenylmethylsulfonyl fluoride, 20 μl complete protease inhibitor (50x), 5 μl RNase inhibitor (40 U/ul). Cell lysis was performed using a covaris sonicator (S220), 10x (10 sec on, 10 sec off, Amplitude 15 μm) until the samples appeared clear. Samples were centrifuged and the supernatant mix with Anti-Flag M2 Affinity Gel (Sigma-Aldrich) beads and incubated overnight at 4 °C on a rotating wheel. After that, the beads were washed 5 times with IP lysis buffer, then eluted in Chip-Seq elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1.0% SDS). Bead pellets were incubated at 65°C in a water bath for 1 hour, then spun at 14,000 rpm for 3 minutes. The supernatant, which contains the Chipped DNA was then collected. Samples were column purified to remove RNA, then fragment ends were repaired and phosphorylated with T4 polymerase and T4 PNK. After that, an A was added to 3' end of repaired ends, then the DNA fragments with 3' A overhang was ligated with illumina TrueSeq Adaptors. Samples were PCR amplified using primers (LC1504_TruSeq_P5 5'- AATGATACGGCGACCACCGAGATCTACAC-'3, LC1505_TruSeq_P7 5'- CAAGCAGAAGACGGCATACGAGAT-'3).