title: GSM2461542: 16698.Scid.adh.2c2_ert2_aH3K4me2_24h_4-OHT-ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-Seq:  Primary cell experiments:  Briefly, lin- FLPs were expanded for 7 days on OP9-DL1, CD25+ enriched and retrovirally infected with empty vector control or HA-tagged versions of Lzr-PU.1WT, -PU.1ENG, -PU.1ETS. Cells were co-cultured on OP9-DL1 for ~40h and then sorted as LiveGFP+CD25+ or LiveGFP+CD44+CD25- on BD FACSAriaTM.    Scid.adh.2c2:    HA-tagged-ChIP: Scid.adh.2c2 cells were infected with empty vector control or HA-tagged versions of Lzr-PU.1WT, -PU.1ENG, -PU.1ETS. Cells were expanded for ~40h and then sorted as LiveGFP+on BD FACSAriaTM.  4-OHT experiment: Scid.adh.2c2 cells were transduced with Lzr-PU.1ert2 (Scid.adh.2c2-PU.1ert2) or Lzr-ert2 (Scid.adh.2c2-ert2) control, cells were expanded for 48h and infected (GFP+) cells followed by sorting of GFP+ cells on BD FACSAriaTM and further expanded in vitro prior to 2, 8 or 24h activation with 0.1 μM 4-OHT (4-hydroxytamoxifen).  Satb1, Runx1, Fog1 and GATA3 ChIP: Scid.adh.2c2 cells were infected with retroviral supernatant of pMxs-Myc-Flag-PU.1-IRES-hNGFR and cultured for 48h.    Approximately 3-5 million primary sorted CD25+ -or 10 million Scid.adh.2c2 cells for ChIP were fixed at RT in 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min followed by additional 10 min after addition of formaldehyde (FA) up to 1% (Satb1, Runx1, Fog1 and Ets1) or 10 min of 1% FA only (PU.1, HA,-tag, GATA3, H3-modifications). The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed in ice-cold 1xHBSS (Gibco). Cell pellets were snap frozen on dry ice and stored in -80°C. For DSG+FA crosslinked cells, nuclei 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 0.5% Lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8)) + PIC. FA crosslinked cells only were resuspended 1% Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8). Lysates were sonicated on a Bioruptor (Diagenode) for 18 cycles, max power for 30s followed by 30 s rest. Sonication was followed by pelleting of debris and the supernatant was transferred to a new tube and chromatin was diluted 3X in 1xHBSS+PIC followed by 2xRIPA (2% Triton, 2mM EDTA, 200 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1% sodium deoxycholate, 0.1%SDS + PIC). Approximately 10 μg per 107 cells of antibody (Rabbit anti-PU.1 polyclonal IgG (sc-352x, Santa Cruz Biotechnology), Rabbit HA-probe polyclonal IgG (sc-805x, Santa Cruz Biotechnology), Rabbit anti-Ets1 polyclonal IgG (sc-350x, Santa Cruz Biotechnology), Mouse anti-Gata3 monoclonal IgG (a mixture of sc-268, Santa Cruz Biotechnology and MAB26051, R&D Systems), Rabbit anti-Satb1 monoclonal IgG (ab109122, Abcam), Rabbit anti-Runx1 polyclonal IgG (ab23980, Abcam), Goat anti-Fog1 polyclonal IgG (sc-9361, Santa Cruz Biotechnolog), Rabbit polyclonal anti-H3K27Ac (ab4729, Abcam), Rabbit polyclonal anti-H3K4me2 (07-030, Millipore) or Rabbit polyclonal anti-H3K27me3 (07-449, Millipore) were hybridized to Dynabeads M-280 Sheep anti- Rabbit or Dynabeads M-280 Sheep anti- Mouse or Dynabeads Protein A/G (Invitrogen), respectively in 1ml 1xRIPA+PIC for 4h, washed twice with 1xRIPA, resuspended in 100 μl 1xRIPA+PIC and added to the diluted chromatin. For polyclonal and monoclonal antibodies, 20 and 50 μl of Dynabeads were used per μg antibody, respectively. ChIP was performed over night at 4°C, and subsequently washed (1 time with 1 ml Low Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton-X, 2 mM EDTA, 50 mM Tris-Hcl pH8, 150 mM NaCl), 1 time with 1 ml High Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton-X, 2 mM EDTA, 50 mM Tris-Hcl pH8, 500 mM NaCl), 1 time with 1 ml LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% Igepal-CA630, 1% natrium deoxycholate, 1 mM EDTA, 10 mM Tris-Hcl pH8), 2 times with 1 ml TE buffer (10 mM Tris–HCl, pH 8.0, 10 mM EDTA)) and eluted for 6 h to O/N at 65°C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 μg proteinase K) treated and finally cleaned up using Zymo ChIP DNA Clean & Concentrator. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (NEB #E6240) following manufacturer’s instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4 and produced approximately 30 million reads per sample. RNA-seq: CD25+ cell experiments:  Briefly, lin- FLPs were expanded for 7 days on OP9-DL1, CD25+ enriched and retrovirally infected with empty vector control or HA-tagged versions of Lzr-PU.1WT, -PU.1ENG, -PU.1ETS. Cells were co-cultured on OP9-DL1 for ~40h and then sorted as LiveGFP+CD25+ or LiveGFP+CD44+CD25- on BD FACSAriaTM.    Scid.adh.2c2: Scid.adh.2c2 cells were transduced with Lzr-PU.1ert2 (Scid.adh.2c2-PU.1ert2) or Lzr-ert2 (Scid.adh.2c2-ert2) control, cells were expanded for 48h and infected (GFP+) cells followed by sorting of GFP+ cells on BD FACSAriaTM and further expanded in vitro prior to 2, 8 or 24h activation with 0.1 μM 4-OHT (4-hydroxytamoxifen).    Total RNA was isolated from 200.000-500.000 primary or Scid.adh.2c2 cells using RNAeasy MicroKit (Qiagen) according to manufacturer’s recommendations. Libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #E7530) from ~1 μg of total RNA following manufacturer’s instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4 and produced approximately 30 million reads per sample. ATAC-seq: Eighty thousand Scid.adh.2c2 or Scid.adh.2c2 transduced Lzr-PU.1-ert2 or Lzr-er2 control vector followed by 0.1 uM 4-hydroxytamoxifen stimulation, 18.4 thousand primary sorted thymic ETP or 50,000 thymic DN3 cells were washed in ice cold HBSS-HEPES prior to the assay. For the primary cells, the final amplified libraries were purified and size-selected (x1.2 bead-to-DNA ratio) using SPRIselect-beads (Beckman-Coultier). Libraries were single-end sequenced on a HiSeq2500 (Illumina) (primary cells) or a NextSeq500 (Scid.adh.2c2) and produced approximately 30-50 million reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM2461542