was exchanged every two days by collecting EBs via gravity- induced sedimentation in 15 ml conical tubes before aspirating spent media, replenishing with fresh media and returning the cultures to the rotary orbital shakers. RNA extraction and quantitative RT–PCR Total RNA from ESCs and embryos was extracted with Trizol reagent (Invitrogen) and Allprep DNA/RNA Mini kit (Qiagen) respectively according to the manufacturer’s instructions. RNA was reverse transcribed using a SuperScript III First-strand cDNA synthesis kit (Life Technologies). Real-time quantitative PCR (qPCR) were performed using iQ SYBR Green Supermix with MyIQ Single Color real-time PCR Detection System (Bio-Rad). The following primers were used: Oct4: forward 59-GCTCA CCCTGGGCGTTCTC-39, reverse 59-GGCCGCAGCTTACA- CATGTTC-39; Nanog: forward 59-CCTCCAGCAGATGCAA- GAACTC-39, reverse 59-CTTCAACCACTGGT TTTTCTG- CC-39; Nkx2.5: forward 59-CAAGTGCTCTCCTGCTTTCC-39, reverse 59-GGCTTTGTCCAGCTCCACT-39; alpha-MHC: for- ward 59-GGTCCACATTCTTCA GGATTCTC-39, reverse 59- GCGTTCCTTCTCTGACTTTCG-39; Tyrosine hydroxylase: for- ward 59-GATTGCAGAGATTGCCTTCC-39, reverse 59-GGG- TAGCATAGAGG CCCTTC-39; Nestin: forward 59-GCCTA- TAGTTCAACGCCCCC-39, reverse 59-AGAC AGGCAGGGC- TAGCAAG-39; AFP: forward 59-AAACTCGCTGGAGTGTCT- GC-39, reverse 59-AGGTTTGACGCCATTCTCTG-39; GFAP: forward 59-GCCACCAGT AACATGCAAGA-39, reverse 59-G- GCGATAGTCGTTAGCTTCG; GAPDH: forward 59-TTCAC- CACCATGGAGAAGGC-39, reverse 59-GGCATGGACTGTG- GTCATGA-39. PCR SuperArray analysis RNA was isolated from ESC and EB samples using QIAshred- ders (as needed) and RNeasy Mini kits (Qiagen) according to the manufacturer’s instructions. RNA quantity and quality were assessed by taking absorbance measurements at 260 and 280 nm on a NanoDrop ND1000 Spectrophotometer (Nanodrop Tech- nologies). First strand cDNA synthesis was performed using the RT2 First Strand Kit (SABiosciences) with 1 mg of input RNA per well followed by real-time PCR using the Mouse Embryonic Stem Cells PCR SuperArray and SYBR Green RT2 qPCR Master Mix (SABiosciences), per manufacturer’s recommended protocols. First strand synthesis and real-time PCR were performed using a BioRad MyCycler and BioRad MyIQ real time thermal cycler, respectively. Array results were first internally normalized to GAPDH levels and subsequently analyzed with Genesis software (Graz University of Technology) using log2 transformation, mean center gene analysis, and hierarchical clustering. Neural differentiation of ESCs ESCs cultures were trypsinized with 0.25% trypsin-EDTA solution, depleted with feeder cells, and resuspended in differen- tiation media at 56104 cell/ml. Embryoid bodies were formed using hanging drop method by plating 20 ml drops (1000 cells per drop) on the inner side of the lid of 15 cm dishes. The bottom of the 15 cm dishes were filled with sterile water and incubated for 4 days. The neural differentiation protocol for ES cells was adapted from ES-Cult Neural differentiation protocols (StemCell Technol- ogies, Vancouver, Canada). Briefly, four days old EBs were collected from the hanging drops and cultured for additional 2 days in 10 cm petri dishes in the presence of 1 mM all-trans retinoic acid. EBs were subsequently plated at 10 EBs per cm2 in tissue culture plates, coated with poly-L-ornithin and laminin (5 mg/ml), in NeuroCult NSC proliferation medium (StemCell Technologies) Loss of H1 Impairs Stem Cell Differentiation supplemented with FGF-b 10 ng/ml. The plates were incubated and the media was change every 2–3 days. Immunocytochemistry Cells grown on glass cover slips were fixed with 4% paraformaldehyde for 20 min at room temperature before immunofluorescence staining. For immunocytochemistry, we used the following primary antibodies: GFAP (Abcam; rabbit IgG; 1:1000), TUBB3 (Millipore; mouse IgG1; 1:50); and secondary antibodies from Molecular Probes or Jackson Immuno Research Laboratories: Cy3-coupled donkey anti-rabbit, Alexa Fluor 488- coupled donkey anti-mouse antibodies. Nuclei were counter stained with Hoechst (1:1000). Images were collected at 206 and 606 on an Olympus Fluorescence Microscope. Preparation and analysis of nuclei and histones of ESCs and EBs mESC and EB nuclei and histones were prepared according to protocols described previously [10,22,23]. Briefly, cultured ESCs or EBs were harvested and nuclei were extracted using 0.5% Nonidet P-40 in RSB (10 mM NaCl, 3 mM MgCl2, 10 mM Tris- HCl, pH 7.5, protease inhibitors) and a Dounce homogenizer at 4uC. Released nuclei were pelleted and resuspended in RSB. Chromatin and histone proteins were subsequently extracted as described previously [22,23]. 50–100 mg of total histone prepara- tions were injected into a C18 reverse phase column (Vydac) on an A¨KTA UPC10 system (GE Healthcare). The effluent from the column was monitored at 214 nm (A214), and the peaks areas were recorded and determined with A¨KTA UNICORN 5.11 software. Relative amounts of total H1s were determined by ratio of the total A214 of all H1 peaks to half of the A214 of H2B peak. The A214 values of the H1 and H2B peaks were adjusted to account for the differences in the number of peptide bonds in each H1 subtype and H2B. Fractions corresponding to the H1d/H1e peak from HPLC analysis were collected and subjected to mass spectrometry analysis on a Qstar XL MS/MS system (Applied Biosystems) with electrospray ionization (ESI) as the ionization method. Analyst QS software (Applied Biosystems) was used for data acquirement and analysis. Mouse embryo preparation H1c+/2H1d+/2H1e+/2 mice were set up for breeding in the afternoon, and embryos were staged as embryonic day 0.5 (E0.5) postcoitus at noon if a vaginal plug was found in the female in the next morning. The female was euthanized and embryos at E8.5 were dissected from the euthanized females according to procedures approved by Institutional Animal Care and Use Committee. DNA and RNA were extracted from embryos using Allprep DNA/RNA Micro kit (Qiagen) according to the manufacturer’s instructions. Genotypes of embryos were deter- mined by PCR assays described previously [15,53]. Quantitative chromatin immunoprecipitation (qChIP) ChIP assays were performed as described previously [10] with modifications. Briefly, crosslinked chromatin was sheared to an average DNA fragment size of 200 to 400 bp by sonication. 20 ml of Dynabeads Protein G (Invitrogen) was incubated with 2 mg of antibody for 7 hours in 4uC. After washing three times with 1 ml PBS containing 0.5% BSA, the Dynabeads were then reacted with 40 mg of soluble chromatin overnight in 4uC. Dynabeads were washed five times with Washing Buffer (50 mM HEPES pH 7.6, 1 mM EDTA pH 8.0, 500 mM LiCl, 0.7% Sodium Deoxycho- late, 1% NP-40) and one time with PBS. Protein/DNA complexes PLoS Genetics | www.plosgenetics.org 12 May 2012 | Volume 8 | Issue 5 | e1002691