title: GSM8990933: DriverMap AIR BCR RNA CDR3 VBC-based (mouse, low input); Mus musculus; OTHER
library_construction_protocol: RNA extraction: QIAGEN RNAeasy Micro/Mini Plus Kit for total RNA isolation. DNA extraction: DNeasy Blood & Tissue kit was used for DNA extraction. AIR-RNA Protocol 1. Hybridization of RevGSP with RNA. 50ng or 10ng of RNA were hybridized with mix of Reverse TCR or BCR (C-region) gene-specific primers (GSPs) in 20ul of 1xHyb buffer at 70C, 5 min, 60C for 60 min, cool down to 25C, add 24 ul (1.2V) of SPRI beads, put the test tube in magnetic stand and wash the beads twice with 200ul of 80% ethanol. 2. Rev GSP extension > cDNA synthesis. The washed magnetic beads were resuspended in 22 ul of 1xRT-extension buffer, remove beads in magnetic stand, collect 20.5 ul of supernatant and incubated at 50C for 35 min, and 72C for 10min. 3. Fwd GSP extension. cDNA product (in 20 ul) was extended by adding 20 ul of DNA polymerase master mix with pool of mouse Forward TCR or BCR FR1 or FR3 GSPs, incubated 98C, 1min, 68C, 10 min and treated with 0.5 ul of primer removal enzyme mix (10x) at 37C, 30min, 95C,5min.  4. 1st PCR. FwdGSP-extended cDNA was diluted in PCR master mix (60ul) with universal anchor primers, and anchored cDNA fragments were amplified in 100-ul of Multiplex DNA polymerase reaction mix with universal anchor PCR primers for 17 cycles (50ng RNA), or 20 cycles (10ng RNA).  5. 2nd PCR.  2ul aliquots of 1st PCR were added in 50ul master mix for 2nd PCR step, amplified using unique combination of Illumina's UDP Fwd-P5-Index and UDP Rev-P7-Index primers for 8 cycles, add extra 1 cycle for AIR-TCR and 3 cycles for AIR-mTCR and AIR-mBCR and treated with primer removal enzyme mix (1-ul) at 37C for 30min. PCR products were analyzed in 3% agarose gel and Fragment Analyzer, combined at 1.5x for FR1 products and ½ for 10ng products amount, purified using AMPpure magnetic beads (1.5X volume). The purified cDNA products were quantitated by Qubit fluorescence measurement, and diluted to 10 nM (1.6 ng/ul) for next-generation sequencing using NextSeq500.    AIR-DNA Protocol 1. RevGSP extension. DNA samples(2ug or 0.4ug) were extended with pool of Reverse AIR TCR or BCR J region GSPs in 50 ul of PCR master mix, incubated at 98C, 1min, 68C, 10 min and treated with 0.5 ul of primer removal enzyme mix (10x), at 37C, 20min, 95C,5min.  2. Fwd GSP extension. Rev GSP-extended DNA product (in 50 ul) was extended by adding 2.5ul of pool of Forward AIR TCR or BCR FR3 GSPs, incubated at 98C, 1min, 68C, 10 min and treated with 0.5 ul of primer removal enzyme mix (10x), at 37C, 20min, 95C,5min.  3. 1st PCR. FwdGSP-extended DNA was diluted in PCR master mix (50ul), and anchored cDNA fragments were amplified in 100-ul of Multiplex DNA polymerase reaction mix with universal anchor PCR primers for 22 (TCR-2ug), 25 (TCR-0.4ug), 25 (BCR-2ug) or 28 (BCR-0.4ug)cycles: (98C, 20sec, 65C, 10sec, 72C,30sec).  4. 2nd PCR.  2ul aliquotes of 1st PCR were added in 50ul of master mix for 2nd PCR step (each tube has unique Dual Index primers) and amplified using unique combination of Fwd-P5-Index and Rev-P7-Index for 8 cycles, treated with primer removal enzyme mix (1-ul) at 37C for 30min. PCR products were analyzed in Agarose gel and Fragment analyzer, combined together at 2x (2ug) and 1x (0.4ug), mix with AIR RNA samples and purified by AMPure beads. The purified cDNA products were quantitated by Qubit fluorescence measurement, and diluted to 10 nM (1.6 ng/ul) for next-generation sequencing using NextSeq500.    Further clarification on the library construction can be found on the DriverMap AIR protocol (https://cellecta.com/pages/product-user-manuals).