GSM1666207: GM12878_K27ac_direct_600_Rep2; Homo sapiens; ChIP-Seq
The procedure was different for preparing sonicated chromatin from 100 or 600 cells directly. Cells were counted with a hematocytometer and 100/600 cells were transferred to a 1.5 ml LoBind Eppendorf tube containing 10 µl 10% FBS in PBS. Cells were then cross-linked for 5 min at room temperature by adding 0.625 µl 16% formaldehyde to yield a final concentration of 1%. Cross-linking was quenched by adding 1.25 µl 2.5 M glycine for 5 min at room temperature. The cross-linked sample was then diluted using 120 µl Covaris sonication buffer (to give a total volume of 130 µl) and sonicated with a Covaris E220 sonicator for 8 min with 5% duty, 105 peak incident power and 200 cycles per burst in a Covaris microtube (520045, Covaris). The sonicated lysate was centrifuged at 14000×g for 10 min at 4°C. Sonicated chromatin in the supernatant was transferred to a new 1.5 ml LoBind Eppendorf tube for MOWChIP-Seq. The ChIPed and input DNA was constructed into libraries by using the ThruPLEX FD Kit (Rubicon Genomics, Ann Arbor, MI, USA). During the amplification step, 11cycles was needed to get enough library from input DNA without over-amplification. 12~13 cycles is needed for ChIPed DNA extracted from 10000 cells. 14~15 cycles is normally required for ChIPed DNA from 1000 or less cells.
GEO Accession
GSM1666207