alias: GSM4663541
title: GSM4663541: WT-Tcrb-Rep2; Mus musculus; RNA-Seq
library_construction_protocol: Trizol extraction The 1ug RNA was used for the reverse transcription (RT). The cDNA libraries were performed with oligo(dT)20 primer by 5′Rapid amplification of complementary DNA ends(5'RACE) . During the reverse transcription reaction , A common adaptor (SMART oligonucleotide) was ligated to  the 5'end of each cDNA . The TCR library is then prepared by touchdown PCR amplification using a set of primers: a forward primer to the adaptor and a reverse primer to the constant region of the TCR  . Touchdown PCR was performed under the conditions of 95°C for 3 min, followed by 15 cycles of 98 °C forc15 s, 65 °C(decrease 0.7°C/cycles) for 15 s and 72 °C for 1 min,  20 cycles of 98 °C for 15 s, 60 °C for 15 s and 72 °C for 1 min and one cycle 10 min at 72°C. PCR products, ranged from 300-700bp were gel purified by DNA Gel Purification Kit. Illumina paired end adapters were ligated to TCR libraries, followed by 2rd round PCR with Illumina P5/P7 adaptor primers under 30s at 95°C , 15s at 65 °C, 60s  at 72°C for 10-15 cycles , Plus a final extenaion for 10 min at 72°C. Ampure bead purified PCR product. Finally, the TCR libraries were sequenced on an Illumina HiseqXtenPE150 (2 × 150 nt).
identifiers.namespace: GEO
identifiers.id: GSM4663541