title: pool of nine mice
organism: Mus musculus
description: Protocols: All animal experiments were performed under the guidelines and protocols approved by the Basel-Stadt cantonal veterinary office (Basel-Stadt Kantonales Veterin_ramt Tierversuchsbewilligung #2582). Female BALB/c mice (n=9, Charles Rivers Laboratories) 6 to 8 weeks old were housed under specific pathogen-free conditions and were maintained on a normal chow diet. Nine BALB/c mice (8-10 weeks old, Charles River, specific-pathogen free) were immunized with 50 ug alum-precipitated chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl (NP, NP-CGG, BioCat). Mice were sacrificed 14 days post-immunization (dpi) and their spleens and bone marrow (from femurs and tibia) were harvested. Antibody-secreting CD138-positive cells were enriched from spleen and bone marrow 14 dpi as previously described (Reddy et al., 2007, Nat Biotech) Total RNA extraction from the pooled ASC population was performed using the TRIzol Plus RNA Purification Kit (Life Technologies), according to the manufacturer's protocol and concentration was determined on a Nanodrop 2000c Spectrophotometer. Enriched total RNA was homogeneously mixed, aliquoted and stored at -80 degrees C Direct Addition method: The Illumina TruSeq Universal Adapter constituted the 5' portion of the forward primers, while the IgG-reverse primer contained the Illumina index primer (Index 4 [Replicate DA1] and 5[Replicate DA2]), thereby directly adding Illumina adapter sequences to PCR products (Fig. 1). 10 PCR reactions were run in parallel with the standard PCR conditions mentioned above, which is equivalent to 500 ng total RNA. Thusly prepared libraries were concentrated and purified as stated above.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-02-20T17:01:10Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T17:08:38Z
attributes.tag: External Id
attributes.value: SAMEA2335566
attributes.tag: INSDC center name
attributes.value: ETH_Zurich
attributes.tag: INSDC first public
attributes.value: 2014-02-20T17:01:10Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T17:08:38Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2226:pool of nine mice
attributes.tag: age
attributes.value: 8 week
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: clinical information
attributes.value: Immunized with NP-CGG (chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl)
attributes.tag: common name
attributes.value: house mouse
attributes.tag: organism part
attributes.value: spleen, bone marrow
attributes.tag: sample name
attributes.value: E-MTAB-2226:pool of nine mice
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: specimen with known storage state
attributes.value: fresh specimen
attributes.tag: strain
attributes.value: BALB/c