6 protocols
AccessionType
nucleic acid library construction protocol
Cells were crosslinked with 1% formaldehyde for 10 min at 37◦C; then, crude nuclei were purified. Chromatin was fragmented by sonication with a Bioruptor UCD-300 (Diagenode) to obtain fragments 200–800 bp in length. For each ChIP assay, 2–5 ug of antibodies were added and incubated at 4◦C overnight. ChIP DNA was quantified with a Qubit fluorometer using the Quant-iT dsDNA HS assay kit ChIP-seq libraries were generated following the procedue described in "TELP, a sensitive and versatile library construction method for next-generation sequencing" by Peng X, Wu J, Brunmeir R, Kim SY, Zhang Q, Ding C, Han W, Xie W, Xu F., published in Nucleic Acids Res. 2015 Mar 31;43(6):e35. doi: 10.1093/nar/gku818. Epub 2014 Sep 15.
growth protocol
Growth medium: DMEM (high glucose), 10% Fetalclone III serum, PenStrep; Induction medium (d0-d2): DMEM (high glucose), 10% Fetalclone III serum, PenStrep, 5uM DEX, 0.5mM IBMX, 0.12ug/ml Insulin, 1uM Rosi, 1nM T3; Differentiation medium (d2-d7): DMEM (high glucose), 10% Fetalclone III serum, PenStrep, 0.12ug/ml Insulin, 1uM Rosi, 1nM T3. Before differentiation cells were seeded at 70% confluency and growth medium was supplemented with 8.3nM human recombinant BMP7 for 3 days.
nucleic acid library construction protocol
Total RNA was extracted using Trizol reagent following the manufactorer's instructions and treated with DnaseI RNA-seq libraries were generated by BGI following their standard procedure: mRNAs is enriched by using the oligo (dT) magnetic beads. By using the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp), then the first strand cDNA is synthesized by random hexamer-primer using the mRNA fragments as templates. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with QiaQuick PCR extraction kit and washed with EB buffer for end repair and poly (A) addition. Finally, sequencing adapters are ligated to the fragments. The fragments are purified by Agarose gel electrophoresis and enriched by PCR amplification.
nucleic acid library construction protocol
Total RNA was extracted using Trizol reagent following the manufactorer's instructions and treated with DnaseI RNA-seq libraries were generated following the procedue described in "TELP, a sensitive and versatile library construction method for next-generation sequencing" by Peng X, Wu J, Brunmeir R, Kim SY, Zhang Q, Ding C, Han W, Xie W, Xu F., published in Nucleic Acids Res. 2015 Mar 31;43(6):e35. doi: 10.1093/nar/gku818. Epub 2014 Sep 15.
growth protocol
Growth medium: DMEM (high glucose), 10% BCS, PenStrep; Induction medium (d0-d2): DMEM (high glucose), 10% FBS, PenStrep, 5uM DEX, 0.5mM IBMX, 0.12ug/ml Insulin; Differentiation medium (d2-d7): DMEM (high glucose), 10% FBS, PenStrep, 0.12ug/ml Insulin. Before differentiation cells were seeded at 70% confluency (d-4).
nucleic acid library construction protocol
Cells were crosslinked with 1% formaldehyde for 10 min at 37◦C; then, crude nuclei were purified. Chromatin was fragmented by sonication with a Bioruptor UCD-300 (Diagenode) to obtain fragments 200–800 bp in length. For each ChIP assay, 2–5 ug of antibodies were added and incubated at 4◦C overnight. ChIP DNA was quantified with a Qubit fluorometer using the Quant-iT dsDNA HS assay kit ChIP-seq libraries were generated following the the manufactorer's instructions (Illumina ChIP-SEQ. DNA Sample prep. Kit, #1003473, lot#6330828)