characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-01-28T17:02:37Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:13:33Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2729896
characteristics.characteristic: External Id
characteristics.text: Yale University
characteristics.characteristic: INSDC center name
characteristics.text: 2015-01-28T17:02:37Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:13:33Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS419788
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2381:id2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: Th1-cell
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: We obtained Myosin VI sufficient and deficient mice from the Jackson Laboratory (Bar Harbor, ME), and bred them in accordance with Yale University Institutional Animal Care and Use Committee. CD4+T cells were isolated from the spleens, axillary, submaxillary and inguinal lymph nodes of 6-8 week-old Myosin VI sufficient and deficient mice. This was done by positive selection with MACS CD4 (L3T4) beads (Miltenyi Biotec, Inc.). Naive CD62Lhigh/CD44low/CD4+/Nk.1.1-/CD25- T cells were purified by flow cytometric cell (FACS) sorting. These cells were in vitro differentiated towards the Th1 lineage by plating 1.5x10^6 naÃ¯ve CD4 T cells with plate-bound anti-CD3 (10mg/ml) (clone 145-2C11 clone, American Type Culture Collection) and anti-CD28 (2mg/ml) mouse antibodies (Pharmingen) in complete Clicks medium in the presence of 50 U/ml rhIL-2 (Peprotech), 3.5 ng/ml murine rIL-12 (generous gift from Genetics Institute) and 10mg/ml 11B11 (anti-IL-4). After 4-5 days of in vitro differentiation, the cells were harvested, separated in a Ficoll gradient, and stimulated or not with plate-bound anti-CD3 (10mg/ml) for 1 hour. In vitro differentiated Th1 cells were stimulated or not with plate-bound anti-CD3 (10mg/ml) for 1 hour. We have followed the protocol previously published in Core, L., Waterfall, J. & Lis, J. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845-1848 (2008). Briefly, nuclei from treated cells were isolated and incubated for 5 minutes with nucleotides and bromo-UTP analog. The nascent RNA from the labeling reaction was isolated and subjected to immunoaffinity purification using Br-dUTP antibody (Santa Cruz Biotech). After first round of purification of nascent RNA, the resulting RNA was ligated with 5' small RNA adaptor RNA (Illumina) and subjected to second Br-UTP purififcation. The resulting RNA is futher ligated using 3' adaptor RNA and subjected to third round of purification. The final purified RNA was reverse transcribed and PCR amplified and size selected (150-250bp) using polyacrylamide gel electrophoresis to generate the final sequencing library. A modified small RNA library contruction protocol based on the Illumina Truseq small RNA kit was modified and incorporated with the nucleic acid extraction protocol of this expermient. Please see the nucleic acid extraction protocol for details.
characteristics.characteristic: description
characteristics.text: untreated
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001461
characteristics.characteristic: growth condtiion
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: myosin 6 deficient
characteristics.characteristic: phenotype
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: id2
characteristics.characteristic: title