title: GSM1666199: FL_LSK_K4me3_100_Rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were cross-linked for 5 min with 1% formaldehyde at room temperature prior to quencing with 125mM glycin. Cross-linked cells were sonicated using Covaris S2 sonicator for 14 min with 5% duty, Intensity 3 and Bursts 200. The sonicated lysate was centrifuged at a speed of 14000×g for 10 min under 4°C. The cleared chromatin (10000 cells per 100 µl) in the supernatant was transferred to a new low-bind Eppendorf tube for subsequent microfluidic ChIP. [Isolation protocol] FLs were dissected from E14.5 embryos. Single cell suspensions were prepared by dissociating mechanically and expelling the cells through 40 μm Nylon filter, followed by red blood cell lysis (ACK Lysing Buffer, Lonza). To remove non-specific binding, anti-mouse CD16/CD32 (Fc Block, Biolegend) were added into single cell suspensions and incubated 10 min at 4°C. Next, Cells were stained with a cocktail of antibodies against several surface markers, including lineage markers (Ly-6G/Ly-6C, CD45R/B220, CD3ε, TER-119, CD4, CD8a, and CD19), CD117 (c-Kit), and Ly-6A/E (Sca-1). Cells were first subjected to yield sort for Lineage-Sca-1+c-Kit+ (LSK) and collected into 500µL 1×IMDM+20% FBS in a 12×75-mm polystyrene tube. Collected cells were then performed purity sort using the same gate strategy and sorted into 0.8 mL 1×IMDM+50% FBS in a 1.5 mL DNA LoBind tube. 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.
attributes.tag: GEO Accession
attributes.value: GSM1666199