Live-organism RNA-seq: C. elegans
To prevent RT primer degradation due to repeated freezing and thawing, 0.2 mL tubes (Axygen, PCR-02-L-C) containing 1 L of 12 M RT primer, barcoded oligo-dT28VN primers, flanking 8-bp UMI, 10-bp cell barcode, sequence for TruSeq i5, and PCR handle, were stocked at -80C. Before sampling, the primers were melted on ice and 1.75 L of nuclease free water (UltraPure DNase/RNase-Free Distilled Water, Invitrogen, 10977015) was added. The capillary containing collected subcellular RNA was broken into the 0.2 mL tube containing RT primer, and the 1 L of inner solution of the capillary was collected into the tube using an air duster (ELECOM, AD-ECOMT). 0.5 L of reaction buffer (10xLysis buffer (635013, Takara) and 2U/L RNase Inhibitor (2313 A, Takara)) was quickly added to the tube, and the intracellular product was denatured for 2 min at 72C and then was placed on ice for 2 min. We added 5.75 L of RT mix (1xFirst-Strand Buffer, 4.8 M biotinylated template switching oligonucleotide (TSO, Qiagen), 2 mM dNTP mix, 4 mM DTT, 2 U/L recombinant RNase inhibitor (2313A, Takara), 20 U/L SMARTScribe Reverse Transcriptase (Takara) to 4.25 L of RNA solution and incubated them in a thermal cycler at 42C for 90 min to obtain first-strand cDNA by reverse transcription and then heated at 70C for 10 min to stop the reaction. To remove excess RT primers, we added 0.5 L of 2.5 U/L exonuclease I (2650A, Takara) and incubated them at 37C for 50 min, followed by inactivation, heating at 80C for 20 min. The first-strand cDNA was amplified by PCR in a 25.75 L reaction containing 0.24 M primer2, 1xSeqAmp PCR Buffer and 0.025 U/L SeqAmp DNA Polymerase (Takara) using the following program: 95C for 1 min; 18 cycles of 98C for 10 s and 65C for 30 s; 68C for 4 min; and 72C for 10 min. We purified the mRNA-derived cDNAs by size fractionation using AMPure beads (B23318, Beckman Coulter). We mixed the cDNA product with 0.7 AMPure beads and placed them on the DynaMag- Spin Magnet (Invitrogen) to capture the beads. The magnetic beads were washed twice on a magnetic stand with 0.2 mL of fresh 70% (vol./vol.) ethanol and air-dried for 2.5 min at room temperature. The cDNAs derived from mRNA were eluted with 8.5 L of elution buffer (10 mM Tris-HCl, pH 8.5). For the mRNA-derived cDNA, we examined the yield using a quantitative real-time PCR (qPCR) targeting GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Hs02758991_g1, Thermo Fisher Scientific) or clec-87 (C-type lectin domain-containing protein 87, Ce02412977_g1, Thermo Fisher Scientific). Since each sample contained only a small amount of cDNA below the detection limit following examination, a maximum of 11 samples were mixed in order from the highest to the lowest Ct value of GAPDH or clec-87, avoiding overlapping of the barcode sequence of the RT primer, and concentrated again using 0.7 AMPure beads. We examined the yield, quality, and size distribution respectively using a Qubit 4 Fluorometer (Thermo Fisher Scientific) and an Agilent High Sensitivity DNA Kit using Bioanalyzer 2100 (Agilent). We then performed the tagmentation of 400 pg of cDNA using a Nextera XT DNA Library Prep Kit (Illumina) and PCR with indexing primers. We then cleaned up the PCR products with 0.6 AMPure beads and eluted them with 6.5 L of Resuspension Buffer (RSB, Illumina). We assessed the yield and length of the library using a Qubit dsDNA HS Assay Kit and the Agilent High Sensitivity DNA Kit (Agilent), respectively. Finally, we quantified the library using the KAPA Library Quantification Kits (Roche) according to the manufacturers protocol. The library was sequenced on a MiSeq or HiSeq X (Illumina) instrument with 2 x 75 bp paired-end reads and 2 x 168 bp paired-end reads, respectively.