title: id2
organism: Mus musculus
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-01-28T17:02:37Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T17:13:33Z
attributes.tag: External Id
attributes.value: SAMEA2729896
attributes.tag: INSDC center name
attributes.value: Yale University
attributes.tag: INSDC first public
attributes.value: 2015-01-28T17:02:37Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T17:13:33Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2381:id2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: Th1-cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: growth condtiion
attributes.value: untreated
attributes.tag: phenotype
attributes.value: myosin 6 deficient
attributes.tag: sample name
attributes.value: E-MTAB-2381:id2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: specimen with known storage state
attributes.value: fresh specimen