Additional file 1: Figure S1.

(a) Quantitative reverse transcription (qRT)-PCR analysis of 7SK total RNA levels in two independent experiments in which embryonic stem cell (ESCs) were nucleofected with antisense oligonucleotides (ASOs) targeting 7SK at a position near the 5′ or 3′ end of the RNA (7SK 5′ or 7SK 53′ ASO). Error bars represent standard error of the mean (SEM) for qPCR technical replicates. (b) qRT-PCR analysis of Dll1 total RNA levels when ESCs were nucleofected with 7SK 5′ and 3′ ASOs. ESCs were replated after nucleofection and collected after 6 hours. Error bars represent SEM for qPCR technical replicates. (c) qRT-PCR analysis of 7SK, Dll1, Olig2, and Hexim1 total RNAs in ESCs after switch to 2iLIF media for several passages. (d) qRT-PCR analysis of Pou5f1 mRNA in ESCs 6 hours after nucleofection with 7SK 3′ ASO. ESCs were grown in serum (Ser-Ser) or 2iLIF media (2i-2i), or switched from 2iLIF to serum media after nucleofection (2i-Ser). Error bars represent SEM from two independent experiments. (e) qRT-PCR analysis of Pou5f1 nascent RNA in ESCs 6 hours after nucleofection with 7SK 3′ ASO. Error bars represent SEM from three independent experiments. (f) Sample preparation workflow for directional RNA sequencing (RNA-seq). Mouse ESCs were transfected with ASOs, and total RNA was extracted after 6 hours. Two independent experimental sets were used. Total RNA samples were treated with DNAse and depleted for ribosomal RNAs, but not enriched for polyadenylated RNAs. After RNA fragmentation and 5′ and 3′ end polishing, adapters were ligated to the RNAs, in accordance with the instructions of the TruSeq Small RNA sample prep kit (Illumina). The amplified DNA was clustered and run in an Hi-Seq instrument (Illumina) to obtain single-end reads of 50 nucleotides in length. Bioinformatic analysis was performed as described in the Materials and Methods section. (g) Breakdown of the number of sequenced reads per sample in the directional RNA-seq, including number of reads mapped to the mouse genome.

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Castelo-Branco et al. Genome Biology 2013 14:R98   doi:10.1186/gb-2013-14-9-r98