title: GSM8527504: MAPseq barcodes; Mus musculus; OTHER
library_construction_protocol: Libraries were prepared as described previously and in the MAPseq protocol (DOI: 10.17504/protocols.io.bsm9nc96):156 Tissue was homogenized in 400 μL TRIzol (Thermo Fisher Scientific, 15596026), and then brought to 1 mL total TRIzol. RNA was extracted according to the manufacturer's protocol and dissolved into 13 μL RNAse-free water. To begin reverse transcription, 4 μL of dissolved mRNA mixed in 6 μL nuclease-free water, 1 μL spike-in RNA (salt purification, IDT, GTCATGATCATAATACGACTCACTATAGGGGACGAGCTG TACAAGTAAACGCGTAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNATCAGTCATCGGAGCGGCCGCTACCTAATTGCCGTCGTGAGGTACGACCACCGCTAGCTGTACA, where ATCAGTCA is the barcode tag of the spike-in and N indicates A, C, G, or T. Spike-in RNA was transcribed in vitro by T7 RNA polymerase and diluted into 103 molecules/ul for target sites and 105 molecules/μL for injection sites) (Integrated DNA Technologies), 1 μL of 10 μM RT primer (salt purification, IDT, CTTGGCACCCGAGAATTCCANNNNNNNNNNNNXXXXXXXXTGTACAGCTAGCGGTGGTCG, where N indicates A, C, G, or T and is the UMI and X is the sample identifier barcode) (Integrated DNA Technologies), and 1 μL of 10 uM dNTPs. The mixture of RNAs was denatured at 70° C for 10 minutes and cooled on ice for 5 minutes. To complete the reverse transcription, 1 μL SuperScript IV (Thermo Fisher Scientific, 18090010), 4 μL SuperScript IV buffer, 1 μL 0.1 M DTT, and 1 μL RNAsin Plus (Promega, N2611) were added into the mixture, and incubated for 10 minutes at 55°C and 10 minutes at 80°C. Samples were pooled and cleaned with 1.8X AMPure XP beads per the manufacturer's instructions (Beckman Coulter, A63881). For second strand synthesis, 17 μL cDNA was added to 2.4 μL SuperScript IV buffer, 0.6 μL 0.1M DTT, 5.6 μL second strand kit buffer (Thermo Fisher Scientific, A48571), 0.75 μL 10mM dNTPs, 0.25 μL e. coli DNA ligase, 1 μL DNA polymerase I, and 0.25 μL RNAseH. These were mixed well and incubated for two hours at 16°C, and then 1 μL T4 DNA Polymerase was added and the sample was incubated for 10 minutes again at 16°C. Second-strand products were cleaned with 1.8X AMPure XP beads (Beckman Coulter, A63881). Following second strand synthesis, 1 μL Exonuclease I (New England Biolabs, M0293S) was added per 16 μL bead-purified product, along with 2 μL of Exonuclease I buffer. The reaction was incubated for 1 hour at 37 °C and inactivated at 80 °C for 20 minutes. All injection sites were pooled together, as were all target sites for the following steps. The reaction was then PCR amplified by adding 25 μL Accuprime Buffer (Thermo Fisher Scientific, 12346086), 25 μL 10 μM Nested1st gfpF primer (HPLC purified, IDT, CTGTACAAGTAAACGCGTAATG), 25 μL 10 μM Nested2nd R primer (salt purification, IDT, CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA), 2.5 μL Accuprime Pfx HF enzyme, and 152.5 μL water. The reaction was incubated at 95ºC for 2 minutes, followed by 15 cycles of 95ºC for 15 seconds and 68ºC for 2.5 minutes. The final extension was at 68ºC for 5 minutes. The samples were again exonuclease digested by adding 5 μL Exonuclease I, incubated at 37 °C for 30 minutes, and inactivated at 80 °C for 20 minutes. The products were diluted 10x for the following PCR steps. For the final PCR, 2.5 μL of diluted product from the first PCR reaction was added to 2.5 μL Accuprime buffer, 2.5 μL 10 μM Sol I primer (salt purification, IDT, AATGATACGGCGACCACCGA), 2.5 μL 10 μM Sol II primer (salt purification, IDT, CAAGCAGAAGACGGCATACGA), 0.25 μL Accuprime Pfx HF enzyme (Thermo Fisher Scientific, 12346086), and 14.75 μL water. The final PCR cycles were determined by testing each sample in multiples of two cycles and running the resultant product on a gel. The minimal number of cycles to produce a visible and clean band at 230 bp was used. The reaction was incubated at 95ºC for 2 minutes, followed by the empirically determined number of cycles of 95ºC for 15 seconds and 68ºC for 2.5 minutes. The final extension was at 68ºC for 5 minutes. The product was then cleaned with the Wizard SV Gel and PCR Clean-Up System (Promega, A9282) and eluted into 40 μL per 1 mL of initial pooled PCR product volume. Then, the purified product was loaded onto a 2% agarose gel, and the 230 bp band was cut and purified with a MinElute Gel Extraction Kit (Qiagen, 28604). The 230 bp product was analyzed on a DNA Bioanalyzer Chip (Agilent, 5067-1504) on a Bioanalyzer (Agilent) to confirm the size and concentration for sequencing and then sequenced on a NextSeq 500 (Illumina) on a high-output paired-end 36 bp run (Illumina, 20024906) with the SBS3T sequencing primer (ACACTCTTTCCCTACACGACGCTCTTCCGATCT, Illumina) for paired-end 1 and the Illumina small RNA sequencing primer 2 (CTTGGCACCCGAGAATTCCA, Illumina) for the second paired-end, resulting in 388 million reads passing Illumina standard quality control.