title: GSM2819757: b DObWT m1; Mus musculus; OTHER
library_construction_protocol: T cells were stained and sorted at the National Jewish Health Flow Cytometry Core Facility and stored at -80°C as pellets. RNA was isolated using the RNeasy Kit (Invitrogen). cDNA was made using the SuperScript VILO Kit (Invitrogen). 1st PCR:  The cDNA is used as a template for a PCR reaction which pools all of the positive strand TCR Valpha oligos.   Each PCR reaction uses a Cα primer, negative strand (see Silberman & al. (2016), PNAS 113(38):E5608-17, doi: 10.1073/pnas.1609717113 for primer sequences).  The positive strand oligo is specific for 20 different Vα's (some families require more than one oligo) and adds suitable adapters for either 454 or Ion Torrent sequencers. All PCR products are separated by size on a 1.5% TBE agarose gel and excised from the gel in the range of 250 bp- 500 bp. 2nd PCR: A reaction using 2.5 ul of the pooled bands is prepared.   The positive primer binds to the sequencing adapter.  The negative primer is just 5' of the Cα oligo used in the first PCR and adds a bar code which denotes the cell sample used. Products are run on a 1.5% TBE gel and the wide band (500-700 bp) is excised from the gel and extracted using a Qiaquik kit without heating.
attributes.tag: GEO Accession
attributes.value: GSM2819757