Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: s2 MNase-seq: Cells were grown in Schneider's medium supplemented with 10% heat inactivated FBS at 28C. For fixation, 107 S2 cells were pelleted and rinsed twice in PBS. Cells were crosslinked in 1.1% formaldehyde in PBS for 10 minutes at room temperature, tumbling end over end in a volume of 10 mL. After addition of 50 uL of 2.5M glycine, the cells were tumbled at room temperature for 2 minutes to quench the reaction. Cells were next rinsed twice with cold PBS before pelleting and flash freezing in liquid nitrogen. For MNase digestion, the cell pellet was resuspended in PBS with 0.1% Triton-X100 (PBS-TX). Digestion of 106 cells per titration point took place in a volume of 400uL PBS-TX supplemented with 1mM CaCl2. Either 1.5U, 6.25U, 25U, or 100U of MNase (Worthington Biochemical) were added to pre-warmed cells and incubated at 37C for 3 minutes. Digestion was halted by moving to ice and adding 10 uL of 250 mM EDTA, 250 mM EGTA. Prior to DNA clean-up, the digestions were adjusted to 0.5% SDS and 10 mM Tris pH 8. For DNA cleanup, digestions were incubated with RNase (Roche) for 30 minutes at 37C, with proteinase K (Roche) for 60 minutes at 55C, and incubated at 65C for 60 minutes to reverse crosslinks. This was followed by phenol-chloroform extraction and ethanol precipitation. For size selection, digestion products in a volume of 100 uL of water were incubated for 5 minutes at room temperature with 60uL of Agencourt AMPure XP beads (Beckman Coulter). After bead separation, the supernatant was moved to a new tube and 120uL of new bead suspension was added. DNA was eluted from these beads and used as input into the library preparation protocol described in Bowman et al. (BMC Genomics 2013). For histone ChIP, after halting the MNase digestion, adding EDTA/EGTA, and adding SDS, 135 uL was removed from the 400 uL digestion as an input fraction and kept overnight at 4C. The remainder of each digestion was split in half (135uL each). Each chromatin aliquot was independently adjusted to ChIP buffer conditions in a volume of 500 uL (10 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.5% sarkosyl, 1% Triton X-100, and 1X COMPLETE protease inhibitors (Roche)). After adding 1 mL additional ChIP buffer to each chromatin aliquot (total volume of 1.5ml), this was tumbled end over end for 10 min at 4C and then subjected to a high-speed spin in a microcentrifuge for 10 min at 4C. Supernatant was taken to a new tube for precipitation. 2 uL of antibody was added to each tube (histone H3 ChIP, ab1791, Abcam). s2 ChIP-seq: After halting the MNase digestion, adding EDTA/EGTA, and adding SDS, 135 uL was removed from the 400 uL digestion as an input fraction and kept overnight at 4C. The remainder of each digestion was split in half (135uL each). Each chromatin aliquot was independently adjusted to ChIP buffer conditions in a volume of 500 uL (10 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.5% sarkosyl, 1% Triton X-100, and 1X COMPLETE protease inhibitors (Roche)). After adding 1 mL additional ChIP buffer to each chromatin aliquot (total volume of 1.5ml), this was tumbled end over end for 10 min at 4C and then subjected to a high-speed spin in a microcentrifuge for 10 min at 4C. Supernatant was taken to a new tube for precipitation. 2 uL of antibody was added to each tube (histone H3 ChIP, ab1791, Abcam; histone H4 ChIP, ab10158, Abcam). esc and npc MNase-seq: J1 ESCs (ESC1) and E14-derived ESCs (ESC2) were maintained on mitomycin-C inactivated embryonic fibroblast feeder layers in DMEM supplemented with 15% fetal bovine serum (Hyclone) and 1000U/ml of leukemia inhibitory factor (EMD Millipore). ESC cultures were depleted of feeder cells prior to use in experiments. Neural progenitors (NPC1) were derived by in vitro differentiation from J1 ESC and maintained using previously described methods38. Embryonic NPCs (NPC2) were isolated from the brains of E13.5 embryos and maintained on poly-l-ornithine coated plates in DMEM:F12 containing B-27 and N-2 supplements (Gibco) and EGF+FGF-2 (10ng/ml). One million Cells were cross-linked at room temperature for 10 minutes with 1% formaldehyde and cross-linking stopped by the addition of 125mM glycine. Nuclei were isolated and MNase digestion performed on either 200,000 or 250,000 nuclei per reaction. Digestions were performed using 64U, 16U, 4U or 1U MNase (Worthington) in 10mM Tris pH 7.4, 15mM NaCl, 60mM KCl, 1mM CaCl2 for 10 minutes (250,000 nuclei reactions) or 15 minutes (200,000 nuclei reactions) at 25C. Reactions were stopped with EDTA/EGTA and 0.5% SDS and 125mM NaCl added to the samples. RNase A and proteinase K treatment along with cross-link reversal at 65C were performed. DNA was purified by phenol-chloroform extraction and column purification. MNase digestion was evaluated and the recovered DNA from 1 -64U MNase digested samples was used to generate sequencing libraries k562 Mnase-seq: Human K562 cells were grown in Iscove's Modified Dulbecco's Medium supplemented with 10% heat inactivated FBS at 37C. Human cells were expanded to yield approximately 4 million cells/reaction and cross-linked with 1.1% formaldehyde for 10 minutes at room temperature. Cells were lysed and nuclei were isolated using a sucrose cushion and treated with a range of 18 MNase concentrations in buffer containing 10mM Tris pH 7.4, 15mM NaCl, 60mM KCl and protease inhibitors for 15 minutes at room temperature. EDTA and EGTA were added to stop the digestion. Cross-link reversal was performed at 65 degrees C for 16 hours followed by RNase treatment for 30 minutes at 37 degrees C, followed by the addition of SDS to a final concentration of 1% and proteinase K digestion overnight, at 55C. DNA was purified by phenol- chloroform extraction and ethanol precipitation. Ampure SPRI beads (Beckman Coulter) were used in a double size selection with ratios of 0.7X and 1.7X to obtain a range of fragment sizes from approximately 100 bp to 1000 bp. s2 RNA-seq: 107 Drosophila S2 cells were harvested on ice, washed in cold PBS and their RNA was extracted using the RNeasy Kit (Qiagen) with on column DNAse digestion according to the manufacturer's protocol. Ribosomal RNA was depleted from the total RNA (5ug) by using the RiboZero gold magnetic kit (Epicentre/Illumina) according to manufacturer's instructions. cDNA was generated with the TruSeq non–stranded kit (Illumina) according to manufacturer's instructions. esc2 and npc2 RNA-seq protocol: mESC and NPC were harvested and ESC were depleted of feeders. RNA was extracted from whole cells using Trizol (Thermo Fisher Cat. No. 15596026). RNA was treated with DNase (Promega Cat. No. M6101) at 37 deg C for 30 minutes. Ribosomal RNA was depleted from the total RNA (5ug) by using the RiboZero gold magnetic kit (Epicentre/Illumina) according to manufacturer's instructions. cDNA was generated with the TruSeq non–stranded kit (Illumina) according to manufacturer's instructions. All libraries were prepared as described in Bowman et al. (BMC Genomics 2013)