title: GSM8251661: OoMII_Nrpp_3; Mus musculus; RNA-Seq
library_construction_protocol: Extraction and library preparation were performed in one reaction (see below) The protocol employed is derived from FLASH-seq (V. Hahaut et al., “Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq.” Nat Biotechnol 40, 1447-1451; 2022), with two main modifications: 1/ Genotyping primers are added during the cDNA amplification step; 2/ A phosphate group is added to the 3' of the template switching primer to exclude the possibility it might prime DNA synthesis during the cDNA amplification step, which would result in tagging single transcripts with multiple UMI barcodes. Embryos were isolated and directly transferred by mouth pipetting to PCR tubes in 5ul of the following lysis solution: 0.1   μL Triton X-100 (10% v/v, Sigma-Aldrich, T8787); 1.2   μL dNTP mix (25 mM each, ThermoFisher Scientific, R1121); 0.46 μL 5'Bio-AAGCAGTGGTATCAACGCAGAGTACT30VN-3', 100 μM (supplied by IDT). 0.46 μL 5' Biotin-AGTGGTATCAACGCAGAGTAC G ATC NNNNNNNN rGrGrG-3′Phospate, 100 μM (supplied by IDT); 0.15 μL recombinant RNase Inhibitor (40 U/μL, ThermoFisher Scientific, AM2684); 0.06 μL Dithiothreitol (DTT, 100 mM; part of the Superscript IV kit, ThermoFisher Scientific, 18091050); 1       μL betaine (5M, Sigma-Aldrich, 61962-50G); 0.45 μL dCTP (100 mM, Sigma-Aldrich, DNTP100); Water to 5 μL final. Notes (AGTGGTATCAACGCAGAGTAC is an suppression PCR sequence. Random Ns form the UMI tag, and ATC is a 3bp barcode that can be varied if multiple samples are to be mixed before tagmentation, and only UMI reads are considered. rGrGrG are required for template switching. The 3' Phosphate inhibits priming in following steps). Tubes were incubated 3 min at 72C in a thermocycler, and immediately transferred on ice. RT-pcr was performed by adding 20ul of the following mix to each sample:1.19 μL Dithiothreitol (DTT, 100 mM; part of the Superscript IV kit, ThermoFisher Scientific, 18091050); 4 μL betaine (5M, Sigma-Aldrich, 61962-50G); 0.23 μL magnesium chloride (1 M, ThermoFisher Scientific, AM9530G); 0.48 μL recombinant RNase Inhibitor (40 U/μL, ThermoFisher Scientific, AM2684); 0.25 uL Superscript IV Reverse Transcriptase (200 U/μL, ThermoFisher Scientific, 18091050); 12.5 μL KAPA HiFi Hot-Start ReadyMix (2x, Roche; cat. KK2502); Water to a final volume of 20 μL. Samples were incubated in a thermocycler set to execute the following programme: Hold at 50°C, and after placing tubes skip to 60 min at 50°C; 3 min at 90°C; Hold at 4°C. Tubes were transferred on ice and 0.5ul of a 100 uM solution of amplification primer (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGCAGTGGTATCAACGCAGAGTAC G) were added to each sample. 1ul of a solution of genotyping primers (see embryo genotyping section) at 10uM each was also added in each tube. Tubes were then transferred to a thermocycler, set to the following programme: Hold at 4°C and after placing tubes skip to 20 sec 98°C; 20 sec 64°C; 6 min 72°C; Followed by 20 cycles of: (20 sec 98°C; 20 sec 67°C ;6 min 72°C, adding 12 seconds per cycle); then hold at 4°C. Amplification products were purified using 1x volume of SPRI magnetic beads, eluting in 15 ul water. 1ul was set aside to perform a nested PCR for genotyping. Fragment size distribution was checked on a Genomic DNA Screentpape (Agilent Technologies, 5067-5365/6), with a typical lengths ranging from less than 1Kb to more than 10kb. For tagmentation, each sample was diluted to 150 pg/ul. 2 ul of diluted cDNA were mixed with 2ul of Amplicon Tagment Mix (ATM) and 4 μL of Tagment DNA Buffer (TD) from an Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024), and incubated for 8 min at 55°C, in a thermocycler. Tn5 was released from DNA by adding 2μL of Neutralization Buffer (NT), followed by a 5 min RT. Enrichment of UMI containing fragments in the tagmented DNA was performed by adding 5 μL of Nextera PCR Master Mix (NPM) and 1ul of 5 uM amplification primer (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG AAGCAGTGGTATCAACGCAGAGTAC G, as before). KAPA HiFi Hot-Start ReadyMix (2x, Roche; cat. KK2502) can substitute the Nextera PCR Master Mix, adapting volumes as required. Tubes were incubated as follows: 72°C for 3 min; 95°C for 30 sec; followed by 10 cycles of linear enrichment PCR: (95°C for 10 sec; 55°C for 30 sec; 72°C for 30 sec); then hold at 4°C. 4 ul of a 2.5uM solution of each of the following amplification primers were added on ice: Universal primer AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG; Indexed primer CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG. The 8 Ns are to be replaced with the Illumina index sequence of choice. Tubes were returned to the thermocycler held at 4°C and then subjected to 14 iterations of the cycle:( 95°C for 10 sec; 55°C for 30 sec; 72°C for 30 sec); Followed by 72°C for 5 mi and then hold at 4°C. Amplification products were purified using 1-1.4x volume of SPRI magnetic beads, eluting in 30 ul water. Fragment size distribution was checked on a DNA Hi Sensitivity D1000 HS Screentape (Agilent Technologies, 5067-5584/5).