To prepare Dicer null ES cells for transfection, feeder cells were removed and 1,500,000 ES cells were seeded on gelatin-coated 6 well plate 1day before transfection. The 10ug plasmids (either wild-type Dicer-pCK or 5' pocket mutant Dicer-pCK) and 10ul Lipofectamine 2000 (Invitrogen) were used for transfection. RNA was extracted at 48hr after transfection and used for preparation of small RNA cDNA libraries. In the case of R1 cells, feeder cells were also removed and 300,000 R1 cells were seeded onto gelatin-coated 60mm-dish. The 30 nM siRNA against GFP and Lipofectamine 2000 (Invitrogen) were used for the transfection. RNA was extracted at 48hr after transfection and used for preparation of small RNA cDNA libraries.
Growth protocol
Dicer null mES (a gift from G. Hannon) was grown on a feeder layer of mouse CF-1 cells or on gelatin coated dishes with knockout DMEM (Gibco, Invitrogen) supplemented with 15% fetal bovine serum (Gibco, Invitrogen), nonessential amino acids (Gibco, Invitrogen), 2mM L-Glutamine (Sigma), 0.1mM 2-mercaptoethanol (Sigma), and 1000unit/ml leukemia inhibitory factor (Chemicon). Another mES cell line R1 was grown on a feeder layer of mouse CF-1 cells or on gelatin coated dishes with GlutaMAX DMEM (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 100 unit/mL penicillin, 100 µg/mL streptomycin (WelGENE), nonessential amino acids (Gibco, Invitrogen), 100 µM 2-mercaptoethanol (Amresco), and 10ng/mL leukemia inhibitory factor (Sigma).
Extracted molecule
total RNA
Extraction protocol
To prepare small RNA cDNA libraries, total RNAs were separated on 15% urea-PAGE and 17~26nt RNAs were gel-purified. Size fractionized RNAs were ligated with 3' adapter using T4 RNA ligase2 trucated form (NEB). After gel purification, 3' adapter-ligated RNAs were ligated with 5' adapter using T4 RNA ligase1 (NEB). The gel-purified ligation products were reverse transcribed using superscript II (Invitrogen) and the cDNA was PCR-amplified with Phusion DNA polymerase (NEB).
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
Illumina Genome Analyzer II
Data processing
All small RNA sequences except low-quality reads are provided with the number of reads for each unique sequence. Illumina SRA v1.5 3' adapter sequences are trimmed using half-open Smith-Waterman algorithm (score parameters: 2 for match, -2 for mismatch, -3 for linear gap penalty). Software used in the data processing is provided in the paper (Park et al. 2011).
