title: GSM1654606: NELF-B ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: primary mammary epithelial isolation protocol: Thoracic and inguinal mammary glands were isolated from 6-8 week virgin mice. Lymph nodes were removed from the inguinal glands. Tissues were quickly minced with scissors and digested with Collagenase and Hyaluronidase for 45 min with gentle shaking. Samples were vortexed vigorously for 15 sec every 15 min during the digestion.  After tissue digestion mammary organoids were collected and red blood cells were lysed. Organoids were further digested by Trypsin and Dispase, and single mammary epithelial cells were obtained after passing through cell strainer. Sonicated (ChIP-seq) or digested (DRIP-seq) genomic DNA was immunoprecipitated by specific antibodies. ChIP-seq and DRIP-seq libraries were built following the instruction of MicroPlex library preparation kit (Diagenode, C05010011).  For RNAPII ChIP-seq, 1 ng of ChIP DNA was used for a total of 15 cycles of PCR amplification.  For NELF-A/B ChIP-seq, 0.2 ng ChIP DNA were used for a total of 18 cycles of PCR amplification.  For DRIP-seq, a total of 15 cycles of PCR amplification was performed. After amplification, libraries were purified using Agencourt AMPure XP system (Beckman Coulter, A63880) following the product manual.  Quantity of the libraries was measured with Qubit dsDNA HS Assay Kit (Life Technologies, Q32851), and quality of the libraries was verified using Bioanalyzer 2100.
attributes.tag: GEO Accession
attributes.value: GSM1654606