title: GSM6614202: retina barcodes rep2 [scMPPRA]; Mus musculus; OTHER
library_construction_protocol: To quantify the cBC-rBC pairs, we performed separate PCRs using primers specifically targeting the reporter gene to improve barcode recovery efficiencies. Because the 10x protocol only uses 1/4 of the generated cDNA, we separately amplified the barcodes from another 1/4 of the pellet cleanup. We first used Q5 (New England BioLabs M0515) polymerase to amplify the region containing the cBC-rBC pairs with SCARED P17 and SCARED P18 with 10 cycles. The sample was divided equally into eight PCR reactions, each with 50 μl of total volume to reduce possible jackpotting. The product was then purified with the Monarch PCR&DNA Cleanup kit (New England BioLabs T1030L), and eluted with 20 μl of ddH2O. We then added sequencing adapters using an additional two rounds of PCR. The first adapter PCR was performed with SCARED P21 and SCARED PP2 with a total of 10 ng of product from the barcode PCR. Again, we pooled eight PCR reactions, each with 50 μl of total volume and 10 PCR cycles. The PCR product was purified using the Monarch PCR&DNA Cleanup kit (New England BioLabs T1030L). For the last PCR, to add the P5 and P7 Illumina adapters, we used the primers SCARED P45, SCARED PP3 with 10ng of product and pooled eight PCR reactions, each with 50 μl of total volume and 10 PCR cycles. scRNA-seq and scMPRA are extracted using 10X protocol