Illumina MiSeq paired end sequencing of SAMD01775584
Total RNA was extracted from FACS-sorted thymocytes using Trizol (Invitrogen). cDNA was synthesized with Superscript III reverse transcriptase (Invitrogen) using a BSL-18E polyT18 primer containing a SphI site. Double-stranded cDNA was generated with E. coli DNA polymerase I, E. coli DNA ligase, and RNase H (all from Invitrogen), followed by blunting with T4 DNA polymerase (Invitrogen). A P10EA/P20EA adaptor was ligated to the 5' end of the ds-cDNA and cut with SphI. After cleanup (Qiagen MinElute Reaction Cleanup kit), primary PCR was performed with KAPA HiFi DNA polymerase (Kapa Biosystems) using TCR alpha constant region-specific primer mCA1 and P20EA. Secondary PCR was performed with mCA2 and P20EA. Amplicons were generated by amplification of the second PCR products using P22EA-ST1-R and mCA-ST1-R. Indexing was performed with the Nextera XT Index Kit v2 (Illumina). Indexed libraries were pooled at equimolar concentrations and sequenced on the Illumina MiSeq platform with 300-bp paired-end reads.