title: GSM5819707: JT_672_Bl6SpOv_E4.5Blastocysts55x_CR_0.1perPFA_Gata6; Mus musculus; OTHER
library_construction_protocol: Cells were harvested using accutase and 100,000 cells per replicate, per time point, were subjected to CUT&TAG and CUT&RUN. Blastocysts were fixed in 0.1% PFA and subjected to CUT&RUN by physically transferring the embryos into different CUT&RUN buffers under a dissection microscope. DNA was issolated using phenol-chloroform extraction. For each sample, double stranded DNA (10µl), Template Preparation D Buffer (2µl), and Template Preparation D Enzyme (1µl) were combined and End Repair and A-tailing was performed in a Thermocycler with a heated lid (22ºC, 25 min; 55ºC, 20 min). Library Synthesis D Buffer (1µl) and Library Synthesis D Enzyme (1µl) were subsequently added, and library synthesis was performed (22ºC, 40min). Immediately after, Library Amplification D Buffer (25µl), Library Amplification D Enzyme (1µl), Nuclease-free water (4µl), and a unique Illumina-compatible indexed primer (5µl) were added. Library amplification was performed using the following cycling conditions: 72ºC for 3 min; 85ºC for 2 min; 98ºC for 2 min (denaturation); 4 cycles of 98ºC for 20 s, 67ºC for 20 s, 72ºC for 10 s (addition of indexes); 14 cycles of 98ºC for 20 s, 72ºC for 10 s (library amplification). Post-PCR clean-up was performed on amplified libraries with a SPRIselect bead 0.6X left/1x right double size selection then washed twice gently in 80% ethanol and eluted in 10-12µl 10 mM Tris pH 8.0. For CUT&TAG, DNA (20µl) was mixed with 5X Q5 reaction buffer (10µl), Q5 polymerase (0.5µl), dNTPs (1µl), nuclease free water (16µl), and an equimolar mixture of an universal i5 and a uniquely barcoded i7 primer (2.5µl) , using different barcodes for each sample. The sample was placed in a Thermocycler with a heated lid using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s and 63 °C for 30 s; final extension at 72 °C for 1 min and hold at 8 °C.