title: GSM2453220: C127_RNAseq rep1; Mus musculus; OTHER
library_construction_protocol: For each individual Bio-CAP experiment, 25 μl of NeutrAvidin Agarose Resin (Thermo Scientific, 29200) was washed with BC100 buffer and then incubated with 50 μl of 0.5 μg/μl biotinylated hKDM2b-CxxC protein diluted in BC100 buffer for 1 h at 4°C. The conjugated resin/CxxC protein was then washed with CAP100 buffer (12.5% glycerol, 0.1% Triton-x-100, 20 mM HEPES pH 7.9, 100 mM NaCl). Genomic DNA was sonicated to an average size of 150-250 bp using a Diagenode Bioruptor. Sonicated DNA was then diluted in CAP100 buffer to a concentration of 16 μg/ml and a 100 μl Input sample was retained. For each Bio-CAP experiment, 500 μl of diluted sonicated DNA, corresponding to approximately 8 μg of DNA, was added to the conjugated CxxC resin. The DNA and resin were incubated at 4°C for 1 h with gentle mixing. The resin was then collected by centrifugation at 1500 g for 3 min at 4°C and the unbound flowthrough (FT) material was removed. The resin and any associated DNA was washed twice with 1 ml of CAP100 buffer, before the first elution was performed by adding 50 μl of CAP300 (12.5% glycerol, 0.1% Triton-x-100, 20 mM HEPES pH 7.9, 300 mM NaCl) to the resin and incubating at room temperature for 10 min. Following centrifugation a 50 μl elution fraction was carefully collected. The elution process was repeated using another 50 μl of CAP300 and the 300 mM elution fractions were pooled (giving a total volume of 100 μl). Subsequent elutions were performed in the same way using buffers with 500 mM, 700 mM and 1 M NaCl sequentially. Each 100 μl elution fraction, together with 100 μl of both the Input and FT samples, was purified using a PCR purification column (Qiagen) and DNA was eluted in a volume of 50 μl. The 700 mM and 1 M fractions were pooled for sequencing. Bio-CAP material was quantified using the High Sensitivity Qubit system (Invitrogen) and the fragmentation profile was assessed using a DNA HS Agilent 2200 TapeStation chip.   Chromatin accessibility was assayed using an adaptation of the assay for transposase accessible-chromatin (ATAC)-seq (Buenrostro et al., 2013). Briefly, 5 × 10^6 cells were harvested, washed with PBS and nuclei were isolated using 1 mL HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05 % NP40 (IGEPAL CA630)), 1 mM PMSF, 3 mM DTT)  for 1 min at room temperature. Nuclei were centrifuged at 1,000 × g for 5 min at 4°C, followed by a total of three washes with ice-cold RSB buffer (10mM NaCl, 10mM Tris (pH 7.4), 3mM MgCl2), to remove as much of contaminating cytoplasmic and mitochondrial material as possible. Nuclei were then counted, and 5×10^4 nuclei were resuspended in Tn5 reaction buffer (10mM TAPS, 5mM MgCl2, 10% dimethylformamide) and 2 µl of Tn5 transposase (25 µM) made in house according to the previously described protocol (Picelli et al., 2014). Nuclei were then incubated for 30 min at 37°C, before isolation and purification of tagmented DNA using QiaQuick MinElute columns (Qiagen). To control for sequence bias of the Tn5 transposase, an ATAC “input” sample was generated, by tagmenting genomic DNA from ESCs with Tn5 for 30 min at 55°C.  Nascent RNA sequencing was performed by pulse labelling with 4-thiouridine (4sU) as previously described (Rädle B. et al., 2013). Confluent 15 cm plates were treated with 500 µM 4sU in 10 ml culture medium for 20 min. The 4sU-containing media was then aspirated and immediately replaced with 5 ml TRIZOL (Thermo Fisher Scientific). RNA was isolated by phenol-chloroform extraction using phase lock tubes (Eppendorf) and resuspend in nuclease-free water. Total RNA aliquots were treated with 6U Turbo DNase (Ambion) and incubated at 37°C for 30 min, followed by inactivation of the DNase according to the manufacturer’s instructions. 300 μg of the resulting total RNA were incubated with 2 μg Biotin-HPDP/μg RNA in biotinylation buffer (10 mM Tris-HCl pH 7.4 and 1 mM EDTA) under rotation for 1.5 h at RT. Unincorporated biotin-HPDP was removed by chloroform/isoamylalcohol extraction. To capture biotinylated RNA, μMACS streptavidin beads (Miltenyi) were added to the RNA suspension (1 μl of beads/ μg RNA) and rotated for 15 min at room temperature prior to loading onto μMACS minicolumns. Unlabelled RNA was removed by washing three times with 900 µl of wash buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20) pre-heated at 65°C, then three more times with wash buffer at room temperature. Biotinylated RNA was then eluted in two rounds with 100 mM DTT and purified using an RNeasy miniElute kit (Qiagen). BioCAP libraries were generated by the High-Throughput Sequencing centre at the Wellcome Trust Centre for Human Genetics (Oxford, United Kingdom) according to standard Illumina library generation protocol and sequenced on the Illumina HiSeq2000 platform with 51 bp reads.   ATAC-seq libraries were prepared by PCR amplification using custom made Illumina barcodes previously described (Buenrostro et al., 2013) and the NEBNext® High-Fidelity 2X PCR Master Mix. ATAC-seq libraries were sequenced on Illumina NextSeq500 using 80bp paired-end reads in biological triplicate.  For RNA-seq library generation, nascent RNA samples were initially depleted of ribosomal RNAs using the Low Input RiboMinus Eukaryote System v2 kit (Thermo Fisher). The recovered rRNA-depleted nascent RNA was used to prepare the cDNA libraries with NEBNext Ultra Directional RNA Library Prep Kit for Illumina and subject to sequencing on Illumina NextSeq500 using 80bp paired-end reads in biological triplicate.