GSM7900900: RNA-seq of Dek-KO E14TG2a cells complemented with HA-Dek-WT repeat 2; Mus musculus; RNA-Seq
The CUT&Tag was performed following the protocol provided by the Hieff NGS® G-Type In-Situ DNA Binding Profiling Library Prep Kit (Yeasen). A total of 50,000 cells were harvested and subjected to two rounds of washing using washing buffer (composed of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and 1× Protease inhibitor cocktail). Subsequently, the cells were incubated with activated Concanavalin A coated magnetic beads. To this, 0.5 μg of HA antibody was added and allowed to incubate on a rotating platform at 4 °C for a duration of 16 hours. Following this, 0.5 μL of Goat anti-Rabbit secondary antibody was introduced and incubated for an additional hour. After a thorough washing step, the cells were treated with pA/G-Tn5 transposase at 37°C for 1 hour. Subsequently, DNA extraction was carried out by DNA selection beads Total RNA was harvested using Trizol reagent from 5*10^6 ESCs. To amplify libraries, 16 μL CUT&Tag DNA was mixed with 1 μL of a uniquely barcoded i5, 1 μL a uniquely barcoded i7 primer, using a different barcode for each sample, 7 μL ddH2O, and 25 μL 2× Ultima Amplification Mix, and run the following cycling conditions with lid heat on: 72 °C for 3 min; 95 °C for 30 s; 12 cycles of 95 °C for 10 s, 55 °C for 30 s and 72 °C for 30 s; final extension at 72 °C for 5 min and hold at 4 °C. Post-PCR clean-up was performed by adding 1.2× volume of DNA selection beads (60 μL), and libraries were incubated with beads for 5 min at RT, washed twice gently in 80% ethanol, and eluted in 20 μL EB buffer. RNA libraries were prepared for sequencing using standard