title: brt22_Inputs
design: To quantify barcodes originating from specific input regions, mRNA from these regions was reverse transcribed using the N2c-RT-Index primer incorporating unique region indices. To evaluate template switching artifacts, in vitro transcribed mRNA containing an alternative barcode version (BCV2) was used as a control, including an additional region index. Each reverse transcription reaction included 4 μL of RNA, 1 μL of 10 μM primer, 2 μL of dNTP mix, 0.5 μL of RNase Out inhibitor, 4 μL of 5x RT buffer, 0.5 μL of Maxima H Minus Reverse Transcriptase, and 8 μL of nuclease-free water. The mixture was incubated at 65 ℃ for 5 minutes for annealing, immediately chilled on ice for at least 1 minute. Extension was performed at 50 ℃ for 20 minutes, and the reaction was terminated by heating at 85 ℃ for 10 minutes, after which samples were kept at 4 ℃. Resulting cDNA was purified using DNA clean beads at a 1.0x ratio. For first-round amplification, primers were designed to bind to the sequence close to the 5' part of the barcode. Each first-round amplification tube contained a mixture of 4 μL of cDNA, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 10 μL of PrimeSTAR Max, and volume filled up to 20 μL with nuclease-free water. First-round amplification was carried out at 98 ℃ for 5 minutes, followed by 20 cycles of 98 ℃ for 30 seconds, 58 ℃ for 30 seconds, 72 ℃ for 30 seconds, with a last extension at 72 ℃ for 5 minutes, and finally ended with 4 ℃ forever. PCR products were purified using 1.2x DNA clean beads. Primers used in the first-round amplification contained adaptors for the second-round PCR to incorporate Illumina adapters. Second-round amplification was carried out at 98 ℃ for 5 minutes, followed by 10 cycles of 98 ℃ for 30 seconds, 58 ℃ for 30 seconds, 72 ℃ for 60 seconds, with a last extension at 72 ℃ for 5 minutes, and finally ended with 4 ℃ forever. Products of the second-round PCR were purified using 1x DNA clean beads, and the purified product was ready for sequencing. By sequencing the library, the abundance of each barcode was determined by counting the unique molecular identifiers (UMI) associated with each barcode.