BCR-Seq of mus musculus: adult male splenic B cells
Isolated B cell RNA was reverse transcribed using SSIV VILO , which contains both poly(dT) and random hexamers for priming. Purified cDNA was split in half to amplify the heavy and light chains in separate reactions. The heavy chains were amplified in a first PCR step with 19 forward primers that anneal in the VH framework 1 region and a mixture of mouse IgG, IgM and IgA reverse primers that contain partial adapter sequences. The light chains were first amplified using a mix of 3 forward primers that anneal in the V or V framework 1 region and 2 reverse primers specific to C or C. The first PCR was performed with Q5 polymerase (NEB). The remainder of the adapter sequences were added to the heavy and light chains separately by a two-step PCR reaction with Q5 using the NEBNext index primers (NEB). The heavy and light chains from the same original sample contained the same index.