GSM1316965: siMLL3_2; Mus musculus; RNA-Seq
Libraries were generated from input chromatin or ChIP enriched chromatin according to Illumina's standard protocol but using reagents from other companies: DNA end-repair was performed using the End-It DNA repair kit, Epicentre (cat:ER81050). A-overhang generation was performed uising Klenow from NED (cat: M0212). Adapter ligation was performede using the LigaFast kit from Promega (cat:M8225). LM-PCR was performed using Phusion DNA pol master mix from NEB (cat:F-531S). Very stringent quality controls were applied to the libraries: 1. Enrichment of a given site had to be preserved in the libraries in a manner comparable to the original ChIP. 10-12 positive and negative sites were tested for library preparation. 2. The total yield of the LM-PCR had to be ~500 ng. 3. The size distribution had to be centred around 350 bp for sonicated with a variation no larger than +/- 75 bp. Libraries that did not pass these criteria were considered substandard and were not used for sequencing.  RNA concentrations were measured using a NanoDrop 8000 Spectrophotometer. 1 ug of total RNA was used for the library preparation, using the Illumina TruSeq RNA Sample Prep Kit (polyA selection-based), following the TruSeq RNA Sample Preparation V2 Guide. TruSeq adapters 1, 3, 8, 9, 10, and 11 were used during the ligation step. 15 cycles of PCR were run during the amplification step. The samples were mixed into one pool and run in one 50-nucleotide single-end read Rapid run flow cell with the Illumina HiSeq 2500 sequencer, to generate a total of 288 million reads passing filter.