title: GSM2879295: RNA-seq-FL-ProB-EBF-KO #3; Mus musculus; RNA-Seq
library_construction_protocol: For RNA-seq: Total RNA was isolated by use of RNAeasy Micro Kit (Qiagen, Hilden, Germany) according to  manufacturer's recommendations. For ATAC-seq: Cells were washed in ice cold PBS prior to Assay for Transposase Accessible Chromatin  (ATAC-seq) library preparation as described in (Buenrostro et al. 2013). For ChIP-seq: After cell sorting FL-cells were cultured  on OP9 supplemented with with 10ng/mL KIT ligand, 10ng/mL Fms-like tyrosine kinase 3 ligand (FLT3L), and 10ng/mL Interleukin-7. Cells for transcription factor ChIP were fixed at room temperature with DSG for 30 min followed by 1% formaldehyde/PBS for 10 minutes. For histne midification ChIP-seq samples were fixed at room temperature in 1% formaldehyde/PBS for 10 minutes. Reaction was then quenched by adding glycine to 0.125 M. Cells were washed  pelleted and snap-frozen and stored at -80°C until ready for ChIP or used immediately for ChIP.   Nuclei for ChIP were isolated  by 10 min incubation in Nuclei Isolation buffer (50 mM Tris-pH 8.0, 60 mM KCl, 0.5% NP40) + protease inhibitor cocktail (PIC) (Roche) on ice. Pelleted nuclei were dissolved in Lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8))+ PIC and sonicated on a Bioruptor (Diagenode) max power for 30s followed by 30 s rest. Sonication was followed by pelleting of debris and the supernatant was transferred to new tube and chromatin was diluted 5X in Dilution Buffer (1% Triton, 2mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8) + PIC) for transcription factor ChIP and in HBSS (Lonza, Verviersa, Belgium) +PIC and 2X RIPA buffer (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 2% Triton X-100, 0.1% SDS, 0.2% Sodiumdeoxycholate, 200 mM NaCl + PIC) for histone modification ChIP-seq. Anti-bodies were hybridized to ProteinA or G Dynabeads®  (LifeTechnologies)  and added to the diluted chromatin. ChIP was performed over night at 4°C. and subsequently washed (1 time with 500 μl Low Salt Immune Complex Wash Buffer, 1 time with 200 μl High Salt Immune Complex Wash Buffer, 1 time with 200 μl LiCl Immune Complex Wash Buffer, 2 times with 200 μl TE buffer) and eluted for 4 h at 65°C ( 20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, 100 μg RNase A and 50 μg proteinase K) treated and finally cleaned up using Zymo ChIP DNA Clean & Concentrator before ChIP-seq libary preparation. ATAC-seq: ATAC-seq library preparation was done as described in (Buenrostro et al. 2013) using Transposomes from Nextera DNA library preparation kit (Illumina, San Diego, CA) and custom primers Libraries were run single read on the Illumina NexSeq500. .RNA-seq: Libraries were constructed using NuGEN's  Ovation Ultralow Library systems (NuGEN Technologies, San Calros, CA) and were subsequently subjected to 76 cycles of single read NextSeq500 sequencing (Illumina, San Diego,CA). ChIP-Seq: Libraries were constructed using Illumina Truseq Nano DNA library preparation kit or by ligation of Nextflex Bioo adaptors (Bioo Scientiffic , Austin, TX) following standard procedures. Libraries were run single read on the Illumina NexSeq500.
attributes.tag: GEO Accession
attributes.value: GSM2879295