GSM2487747: neuron_YA_2; Caenorhabditis elegans; RNA-Seq
Total RNA was extracted with the Trizol/phenol/isopropanol method as described (Invitrogen) A total of 300–600 μg RNA was pulled-down with a biotin-labelled probe (antisense to tag, biotin-AGATCGGAAGAGCGTCGTGT) with a PolyATract mRNA kit according to the manufacturer's instruction (Promega, #Z5310) except that the polyT probe in the kit was replaced by the anti-tag probe. All of the pulled-down RNA was then reverse-transcribed with pRT(ATTTAGGTGACACTATAGAAGCAGAAGACGGCATACGAT(20)V) with PrimeScriptTM II Reverse Transcriptase (Takara, #2690A) at 42°C for 90 min to get full length cDNA. Then the cDNAs were PCR amplified for 10 cycles (98°C 10 s, 58°C 30 s, 72°C 1.5 min) with primer pF1 (ACACGACGCTCTTCCGATC) and pR1 (ATTTAGGTGACACTATAGAAGCAGAA) with Ex-Taq HS (takara, #RR006). To get higher specificity, nested PCR was performed (98°C 10 s, 58°C 30 s, 72°C 1.5 min) with pF2 (GACGCTCTTCCGATCTGGTT) and pR2 (CAAGCAGAAGACGGCATACGA) for another 10 cycles. The resulting cDNAs were fragmented and tagged with adapters with Tn5 transposase (Vazyme) at 55°C for 10 min, then amplified with pFY(AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTT) and pN7(CAAGCAGAAGACGGCATACGAGATIIIIIIIIGTCTCGTGGGCTCGG, ‘I’ represents index) with Q5 polymerase (NEB, #M0494) for 12–15 cycles (pre-PCR: 72°C 3min, PCR-cycle: 98°C 10 s, 66°C 30 s, 72°C 50 s) and purified with Ampure XP beads. PCR product was then sequenced on an Illumina HiSeq2000 using 2 × 100 bp or 2 × 125 bp mode.