Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) from formaldehyde-fixed material

In crosslinked MINUTE-ChIP, formaldehyde-fixed chromatin is fragmented using sonication, blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Here, we demonstrate a MINUTE-ChIP for Nanog from formaldehyde-fixed cells using fragmentation by sonication. For fixed anti-Nanog MINUTE-ChIP, Rw4 (129X1/SvJ) mouse embryonic stem cells were cultured in a naive pluripotent state in the presence of LIF (the Nanog-high condition). As a negative control, the cells were cultured in same medium ibut without LIF for 24 hr, then 48h in the presence of 2uM retonoic acid (Sigma R2625), to yield a Nanog negative condition (Nanog is absent by western blot under this condition). 1 Mio cells in were barcoded in duplicates for each condition and pooled.