4 protocols
normalization data transformation protocol
Illumina Offline BaseCaller1.9.3 software used for basecalling. Sequence reads were mapped to mm9 whole genome using Tophat 1.14/ Bowtie 0.12.7 with the following parameters --min-anchor-length 6 --splice-mismatches 0 --min-intron-length 10 --max-intron-length 1000000 --min-isoform-fraction 0.0 --max-multihits 50 --no-novel-juncs --library-type fr-unstranded Gene expression was calculated as Fragments per Kilobase of modeled exon per Million mapped reads (FPKM) using Cufflinks 1.3.0, and transcription start site (TSS)-specific gene expression were obtained from Cuffdiff Genome_build: mm9 Supplementary_files_format_and_content: A tab-delimited text file that includes RPKM values for each Sample, and log2(fold-changes) among samples at each time-point (with an added pseudo-count of 0.01 to each RPKM value).
growth protocol
V6.5 (129SvJae and C57BL/6; male) ESCs were plated with irradiated murine embryonic fibroblasts (MEFs) and grown under typical ESC conditions on gelatinized tissue culture plates. Briefly, cells were grown in Knockout DMEM supplemented with 10% fetal bovine serum, leukemia inhibitory factor (LIF), non-essential amino acids, L-glutamine, and penicillin/streptomycin as previously described (Boyer et al., 2006). ESCs used for the majority experiments, including ChIP-Seq, RNA-Seq and RT-qPCR, were plated without irradiated MEFs for the final passage.
sample treatment protocol
H2AZ-YFP, Acidic Patch (AP3)-YFP and H2A-YFP constructs were generated from the pAd-cDNA (Addgene). The AP3 H2AZ mutant is a triple point mutant of H2AZ in essential acidic patch region generated by replacing the H2AZ residues (G92N, D97N and S98K) in this region with the corresponding H2A residues, with the GFP in this vector being replaced by YFP, such that it is in frame C-terminal to the cDNA. EcoRI and XbaI digestion was performed to place H2AZ, AP3 and H2A cDNA in frame. This vector contains a CMV-promoter driven by an rtTA drug inducible system. The resulting lentiviral constructs were transfected into 293 cells using the protocol outlined by the RNAi consortium (BROAD Institute, http://www.broadinstitute.org/rnai/public/). The viral supernatant generated 48hrs after transfection was used to infect KH2 ESCs (Beard, 2006) to generate wildtype and mutant H2AZ transgenic ESC lines. The YFP transgenic ESCs were induced with 1µg/ml of doxycycline and FACS sorted for YFP positive cells. In order to study the selective impact of the H2AZ mutants, lentiviral constructs expressing short hairpins specifically directed at the 3’ UTR of endogenous H2AZ were introduced into the wild-type and mutant H2AZ transgenic KH2 ESC lines using RNA. Sequences of the different H2AZ 3’UTR-directed hairpin oligos are as follows: sh#2 5’- AACAGCTGTCCAGTGTTGGTG-3’; sh#5 5’- AATTAGCCTTCCAACCAACCA-3’. Hairpin oligos were annealed and cloned into pLKO.1 vector (Sigma) as detailed by the RNAi consortium, BROAD (http://www.broadinstitute.org/rnai/trc/lib). Blasticidin was used as a selection marker for the generation of endogenous H2AZ-depleted transgenic KH2 ESCs. The puromycin marker in the pLKO.1 vector was removed by digestion with BamHI and KpnI and replaced with blasticidin. The blasticidin cDNA was PCR amplified from pLenti6.2/V5-DEST Gateway® Vector (Invitrogen). V6.5 (129SvJae and C57BL/6) and the YFP transgenic ESCs were cultured as previously described (Boyer et al., 2006). The endogenous H2AZ-depleted transgenic KH2 ESCs were similarly cultured with the addition of blasticidin (5µg/ml) on blasticidin-resistant feeder cells (Iuchi, 2006).
nucleic acid library construction protocol
RNA was extracted using TRIzol. The purified RNA was the subjected to oligo (dT) selection, fragmentation and first and double strand synthesis with the Illumina Tru-Seq kit using manufacturer’s instructions. DNA fragments above 30bp was purified using SPRI-TE beads according to manufacturer’s instructions. The purified DNA was end repaired and single A bases were added for adaptor ligations. The adaptor-ligated DNA was then subjected to double SPRI-TE purification to select for ~200bp DNA fragments. These fragments were enriched and barcoded by PCR for multiplexing. A final SPRI-TE purification was performed to clean up the barcoded RNA-Seq libraries.