7 protocols
high throughput sequence alignment protocol
Sequence reads were trimmed to remove any trailing 3'-adapter sequence using Reaper version 12-048 (http://www.ebi.ac.uk/research/enright/software/kraken) with options: -3p-global 12/1/0/2 -3p-prefix 12/1/0/2 -3p-head-to-tail 1. Reads shorter than 20 nt after trimming were discarded. Remaining sequences were aligned to mouse genome assembly NCBIM37 (mm9) using GSNAP version 2012-04-21. GSNAP parameters were set to require 95% similarity and disable partial alignments (-m 0.05 --terminal-threshold=100 --trim-mismatch-score=0). To enhance alignment accuracy, GSNAP was provided with known splice sites from Ensembl 66 and the RefSeq Genes and UCSC Genes tracks from the UCSC Genome Browser database. Reads coinciding with ribosomal RNA transcripts from Ensembl or ribosomal repeats in the UCSC Genome Browser RepeatMasker track were excluded. A UCSC track hub to visualize the data on the mouse genome is provided in an additional file: https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-1585/7SK_track_hub.tar.gz.
nucleic acid sequencing protocol
Sequencing was performed in a HiSeq 2000 instrument (Illumina) to produce 50nt single-end reads.
nucleic acid library construction protocol
Total RNA was depleted from ribosomal RNA with the Low Input Ribo-Zero rRNA removal system (Epicentre). rRNA-depleted samples were then fragmented with RNA fragmentation reagent (Ambion), purified using the RNeasy MinElute Kit (Qiagen) and treated with alkaline phosphatase (New England Biolabs) for 30 minutes at 37 degrees C. 5' dephosphorylated RNA was treated with T4 PNK Kinase (New England Biolabs) for 60 minutes at 37 degrees C. The resulting RNA (5' mono-phosphorylated and 3' OH) was purified with RNeasy MinElute (Qiagen) and ligated with RNA 3' and 5' TruSeq adapters (Illumina). Indexes 1-6 were used for library construction.
nucleic acid extraction protocol
Total RNA was isolated using miRNeasy extraction kit (Qiagen) with in-column DNase treatment.
growth protocol
Oct4-GIP mouse embryonic stem (ES) cells (Ying et al. 2002) were cultured in either standard ES cell medium (GMEM with 10% fetal calf serum (Biosera), 0.1mM non-essential amino acids, 2mM L-Glutamine, 1mM sodium pyruvate, 0.1mM beta-mercaptoethanol and 10^6 units/L LIF (ESGRO, Millipore)), or in 2i/LIF medium (GMEM with 10% Knock-Out Serum Replacement (Life Technologies), 1% fetal calf serum (Biosera or Sigma), 0.1mM non-essential amino acids, 2mM L-Glutamine, sodium pyruvate, 0.1mM beta-mercaptoethanol, 1uM PD0325901 (AxonMedChem), 3uM CHIR99021 (AxonMedChem) and 10^6 units/L LIF (ESGRO, Millipore)). 1ug/ml puromycin was added to Oct4-GIP ES cell cultures during expansion.
treatment protocol
Cells were transfected with 1000 pmol antisense oligonucleotides (Integrated DNA Technologies) using the Mouse ES Cell Nuclefector Kit (Lonza).