The reaction products were purified using the MinElute PCR purification kit (Qiagen, Valencia, CA), separated by 2% agarose gel electrophoresis to separate the library from adapter-adapter ligation products, and purified from the gel using the MinElute gel purification kit (Qiagen, Valencia, CA). The thermocycling parameters were: 95C 2 min, 98C 30 sec, then 7 cycles of 98C 15 sec, 65C 30 sec and 72C 3 min, ending with one 72C 5 min step. One third of the bisulfite-converted, adapter-ligated DNA molecules were enriched by 7 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5ul 10X PfuTurbo reaction buffer, 25uM dNTPs, 0.5uM of Illumina PCR primers. Gel purified DNA was then converted with sodium bisulfite using the Imprint DNA Modification Kit (Sigma-Aldrich) as per manufacturer's instructions. Adapter-ligated DNA of 140-210 bp (for single end sequencing) or 340-410 (for paired end sequencing) was isolated by 2% agarose gel electrophoresis.
Annealed adapters were ligated to the DNA fragments as per manufacturer's instructions for genomic DNA library construction. Adapters were ordered as single stranded oligos (Microsynth AG), resupended in annealing buffer (10mM Tris pH7.5, 50mM NaCl, 1mM EDTA), annealed by heating at 95 degrees C. For single end sequencing: 5' P-GATXGGAAGAGXGGTTXAGXAGGAATGXXGAG and 5' AXAXTXTTTXXXTAXAXGAXGXTCTTXXGATXT, and for paired end sequencing: 5' P-GATXGGAAGAGXGGTTXAGXAGGAATGXXGAG and 5' AXAXTXTTTXXXTAXAXGAXGXTXTTXXGATXT where X is a methylated cytosine. Adapter sequences were reproduced based on Illumina adapter sequences (Oligonucleotide sequences 2006-2008 Illumina, Inc. for 30 minutes with 200uM dATP, 1xNEB Buffer 2, 15 units Klenow Fragment (3'-> 5' exo-)(NEB # M0212L). 3' ends of DNA fragments were adenylated by incubation at 37 degrees C. (Klenow) (NEB #M0210S), 50 units of T4 PNK (NEB #M0201S), 1x T4 DNA ligase buffer containing 10mM ATP (NEB). for 30 minutes with 400uM dNTP, 15 units of T4 DNA polymerase (NEB #M0203S), 5 units of DNA Polymerase I Lg. DNA fragments were end repaired by incubation at 20 degrees C. Briefly, One to five ug of input DNA were fragmented by sonication to 50-500 bp with a Bioruptor (Diagenode, Sparta, NJ). The protocol was adapted from Illumina Genomic DNA Sample Preparation Guide and Paired-End Sample Preparation Guide. Wild-type embryonic stem cells (129-C57Bl/6) were cultured and differentiated as previously described (M.
GEO help: Mouse over screen elements for information.Ĭell type: ES-derived neuronal progenitor cells
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