title: NextSeq 500 sequencing; PRO-seq in control and H3.3 K4A mutant mESCs
design: PRO-seq in control and H3.3 K4A mutant mESCs
library_construction_protocol: Mouse embryonic stem cells (129B-13) were cultured in ESC media containing Knockout-DMEM (Thermo Fisher) with 15% EmbryoMax FBS (Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression Facility at EMBL Heidelberg), 1% non-essential amino acids, 1% Glutamax, 1% Pen/Strep, 1% of 55mM beta-Mercaptoethanol solution. Cells were maintained at 37C with 5% CO2. Precision nuclear run-on transcription sequencing (PRO-Seq) experiments were performed as previously described in Mahat D. B. et al. (2016). In brief, 10 million permeabilized cells  were used for each run-on assay. In vitro nuclear run-on assays were carried out by adding100 ??l of 2?? NRO reaction mixture (10 mM Tris-Cl pH 8.0, 300 mM KCl, 1% Sarkosyl, 5 mM MgCl2, 1 mM DTT, 375 ??M biotin-11-NTPs, and 0.8 u/??l RNase inhibitor) to 100 ??l of the permeabilized cells, and incubated at 30 ??C for 3 minutes. Reaction was terminated by TRIzol (Thermo Fisher Scientific) addition and nuclear RNA was extracted and precipitated in 75% ethanol. Nuclear RNA was fragmented by base hydrolysis in 0.2 N NaOH on ice for 10 minutes and neutralized by adding the same volume of 1 M Tris-HCl pH 6.8. Fragmented biotinylated RNA was bound to the Streptavidin magnetic beads (Thermo Fisher Scientific), washed, eluted using TRIzol, and precipitated in ethanol. Following 3??? RNA adaptor and 5??? RNA adaptor ligation, RNA was reverse transcribed and PCR amplified. DNA libraries were PAGE purified and submitted for sequencing. Libraries were quality controlled using Bioanalyzer and sequenced on the Illumina NextSeq 500.  Precision nuclear run-on transcription sequencing (PRO-Seq) experiments were performed as previously described in Mahat D. B. et al. (2016). In brief, 10 million permeabilized cells were used for each run-on assay. In vitro nuclear run-on assays were carried out by adding100 ??l of 2?? NRO reaction mixture (10 mM Tris-Cl pH 8.0, 300 mM KCl, 1% Sarkosyl, 5 mM MgCl2, 1 mM DTT, 375 ??M biotin-11-NTPs, and 0.8 u/??l RNase inhibitor) to 100 ??l of the permeabilized cells, and incubated at 30 ??C for 3 minutes. Reaction was terminated by TRIzol (Thermo Fisher Scientific) addition and nuclear RNA was extracted and precipitated in 75% ethanol. Nuclear RNA was fragmented by base hydrolysis in 0.2 N NaOH on ice for 10 minutes and neutralized by adding the same volume of 1 M Tris-HCl pH 6.8. Fragmented biotinylated RNA was bound to the Streptavidin magnetic beads (Thermo Fisher Scientific), washed, eluted using TRIzol, and precipitated in ethanol. Following 3??? RNA adaptor and 5??? RNA adaptor ligation, RNA was reverse transcribed and PCR amplified. DNA libraries were PAGE purified and submitted for sequencing. Libraries were quality controlled using Bioanalyzer and sequenced on the Illumina NextSeq 500. Nuclear RNA was fragmented by base hydrolysis in 0.2 N NaOH on ice for 10 minutes and neutralized by adding the same volume of 1 M Tris-HCl pH 6.8. Fragmented biotinylated RNA was bound to the Streptavidin magnetic beads (Thermo Fisher Scientific), washed, eluted using TRIzol, and precipitated in ethanol. Following 3??? RNA adaptor and 5??? RNA adaptor ligation, RNA was reverse transcribed and PCR amplified. DNA libraries were PAGE purified and submitted for sequencing. Libraries were quality controlled using Bioanalyzer and sequenced on the Illumina NextSeq 500.
attributes.tag: Experimental Factor: genotype
attributes.value: H3f3a -/-,H3f3b K4A