GSM4654352: si-RBFOX1, rep1; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy plus mini kit (Qiagen, Courtaboeuf, France). cDNA was prepared from 300 ng total RNA using 2.5 μM oligo dT and Superscript II (Thermo Fisher Scientific) as described by the manufacturer.The full-length DMD cDNA was amplified as a single long fragment (11.3 kb) by using the GoTaq Long PCR Master Mix (Promega, Charbonnières-les-Bains, France) and primers located in exon 1 (5′ -CTTTCCCCCTACAGGACTCAG-3′) and in the 3′ UTR (5′ -CCAAATCATCTGCCATGTGG-3′) of the muscle isoform (Dp427m) ﻿. ﻿LR-PCR reactions were purified using Qiaquick PCR purification kit (Qiagen) and quantified using the NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific) The library was prepared from 700 ng of purified PCR product fragmented by nebulization (2.1 bars of nitrogen for 1 min) and subjected to library preparation with the Rapid Library Preparation Kit (Roche) according to manufacturer's instructions. The different samples were labeled via ligation of Multiplex Identifiers (MID) oligonucleotide adaptors to allow multiplexing. Three Multiplex Identifiers (MID)-tagged samples were pooled for simultaneous amplification and sequencing on the Roche GS Junior 454 sequencer.