title: Illumina HiSeq 1000 paired end sequencing; Nuclear RNA-seq in WT, Ring1a KO and Ring1a/b dKO mouse ES cells
design: Nuclear RNA-seq in WT, Ring1a KO and Ring1a/b dKO mouse ES cells
library_construction_protocol: J1 (129S4/SvJae) and 129SvJae/Cast WT mESCs were grown under standard ESC culture conditions. Ring1A−/−;Ring1Bfl/fl;Rosa26::CreERT2 mESCs (Ring1A-KO) were cultured as described previously in Endoh et al. Dishes were coated with 0.1 % gelatine and irradiated mouse embryonic fibroblasts (MEFs). For harvesting, ESCs were trypsinized and pre-plated twice for 30 min to remove contaminating feeder cells. Mouse ESCs were washed in PBS and approximately 30-50 x 106 ES cells were lysed for 5 min in 0.5 ml cold buffer RLN (50 mM TrisHCl pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.4% Igepal). Nuclei were pelleted by spinning at 300g for 10 min at 4°C. Nuclear RNA was isolated using TRIsure (Bioline), treated with DNaseI (Roche) and re-purified using an RNeasy Mini Kit (Qiagen). Strand-specific RNA-seq libraries were prepared as described previously by marking the second strand with dUTP but with some modifications8,9. 250 ng nuclear RNA was fragmented using a Covaris E220 instrument at standard RNA settings for 60 sec. Fragmented RNA was precipitated and first strand synthesis was carried out using SuperScript III (Invitrogen) with 4 µg of actinomycin D (Sigma). Nucleotides were removed with mini quick spin DNA columns (Roche) and second strand synthesis was performed using E.coli DNA ligase (NEB), DNA Polymerase I (NEB) and RNase H (Fermentas), replacing dTTP with dUTP (Fermentas). Following purification on QIAquick columns (Qiagen), TruSeq Illumina adapters were ligated with T4 DNA Ligase, (Enzymatics). Libraries were purified on QIAquick columns, treated with USER (NEB) to destroy the second strand and size selected using AMPure XP beads. Libraries were amplified with 9-11 PCR cycles.
attributes.tag: Experimental Factor: phenotype
attributes.value: wild type
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype
attributes.tag: LIBRARY_STRAND
attributes.value: first strand