title: GSM1972421: RNA-seq CTL_MB_Rep2; Mus musculus; RNA-Seq
library_construction_protocol: For ChIPSeq, lysates were cleared from sonicated nuclei and used for immunoprecipitation. For RNASeq, RNA was extracted according to TRIzol protocol (invitrogen). For ATACSeq, 5x104 C2C12 cells were pelleted, washed with 50ul of 1xPBS and lysed in 50ul of Lysis Buffer (10mM Tris-HCl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% of IGEPAL CA-630).  To tag and fragment accessible chromatin, nuclei were centrifuged at 500x g for 10min and re-suspended in 40ul of transposition reaction mix with 2ul Tn5 transposase (Illumina Cat# FC-121-1030). The reaction was incubated at 37ºC with shaking at 300rpm for 30min.  DNA-fragments were then purified, and amplified by PCR (12-15 cycles based on the amplification curve). For ChIPSeq, cells were cross-linked in 1% formaldehyde and lysate to extract nuclei. The chromatin was sonicated to 250 bp fragments. Histone-DNA complexes were isolated with anti-histone mH2A1.2 (Cell signaling, 4827S), anti-mH2A1 (Abcam, ab37264), anti-Pbx1 (Abnova, H00005087), anti-histone H3 (acetyl K27) (Abcam, ab4729), anti-trimethyl-histone H3 (Lys27) (Millipore, 07-449). 10ng of immuno-precipitated DNA fragments were used to prepare ChIP-seq libraries with the NEBNext RNA library prep kit (New England BioLabs) and Ovation SP Ultralow DR Multiplex system (Nugen) following the manufacturer’s protocol. For polyA RNA-Seq, Illumina protocol was followed.  ATACSeq samples were multiplexed using primers Ad2.1-4 paired with Ad1 for final library amplification as described previously (Buenrostro et al., 2013).
attributes.tag: GEO Accession
attributes.value: GSM1972421