alias: M_17_5740
title: ChIRP Control MALAT1  Replicate 1
design: Biotin labelled antisense oligo probes were designed against Malat-1 lncRNA and purchased from Sigma. The following conditions were met: 1) 1 probe per 100bp of length; 2) GC% target =45; 3) oligonucleotide length=20; 4) spacing length of 60-80bp; probes were numbered and even probes were pooled together, while the odd pool contained all the odd-numbered probes. The pools were diluted in up to final concentration as 100 M. C2C12 cells were strongly fixed using 1% glutaraldehyde for 10 minutes at room temperature. Then, quenching was done using final concentration 125 mM glycine for 5 minutes at room temperature. The cells were pelleted, lysed and sonicated (BIORUPTOR, 30 seconds ON, 30 seconds OFF and high intensity, for 3-4 hours until the solution became clear. The sonicated samples were centrifuged at 16,000 RCF for 10 min at 4C and aliquoted into 1ml samples per tube. 10L for RNA input and 10 L for DNA input were withdrawn from the samples. All input samples were stored at -20 C for further step. 2 ml of hybridization buffer was added to each 1ml of sample. 1L of 100 pmol/L probes per 1 mL chromatin were added to each sample, which was mixed well and incubated at 37C for 4 hours with Thermomixer shaking. C-1 magnetic beads (Invitrogen) were washed with lysis buffer and 100L of beads per 100 pmol of probes were added to the samples and left for 30 minutes at 37C with shaking. After five rounds of washing, the beads were resuspended with 1ml of wash buffer. 100L was taken for RNA isolation using Trizol out of each 1ml sample. The remaining sample was used to isolate the DNA and library preparation. Library preparation was prepared with MicroPlex Library Preparation Kit v2 (Diagenode). Final library was pooled and sequenced at Illumina HiSeq 4000 platform.
identifiers.namespace: SUB8074832
identifiers.id: M_17_5740