ChIP-Seq_Multiple myeloma_
design_description
NEXTflex ChIP-Seq from Bioo Scientific, 10 cycle PCR at the end and last purification is performed with magnetic beads.
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR
Bioruptor Plus (UCD-300)
CHIP_ANTIBODY_PROVIDER
Diagenode
CHIP_PROTOCOL_BEAD_TYPE
magnetic beads A and G
EXPERIMENT_TYPE
Histone H3K4me3
CHIP_ANTIBODY_LOT
A5051-001P
CHIP_PROTOCOL
Transcription factor protocol: 100.000 cells per ChIP (5x) and pool at the end. Overnight incubation of chromatin with antibody and A/G magnetic beads. Wash twice with buffer 1, once with buffer 2 and 3 and twice with buffer 4 at 4 degrees. Elute 20 min with elution buffer at RT. Descrosslink 4 hours at 65 degrees. Purify by column purification.
CHIP_PROTOCOL_BEAD_AMOUNT
20 µl
CHIP_PROTOCOL_CHROMATIN_AMOUNT
5x 100.000 cells = 5x 6,7 µl
CHIP_PROTOCOL_ANTIBODY_AMOUNT
1,0 µg
EXTRACTION_PROTOCOL
Chromatin was extracted using the histone protocol. Cells are crosslinked 10 min in 1% formaldehyde. Washed twice in PBS and dissolved in shearing buffer (containing 1% SDS), such that the final concentration is 15*10^6 cells/ml.
CHIP_ANTIBODY_CATALOG
pAb-003-050
CHIP_ANTIBODY
H3K4me3
EXTRACTION_PROTOCOL_SONICATION_CYCLES
3x 10 min; 30 sec ON/ 30 sec OFF