title: GSM5421454: sorted ensheathing glia 10E12 d70 rep3; Drosophila melanogaster; RNA-Seq
library_construction_protocol: RNA was purified with TRIzol. Purified RNA in Smart-seq3 buffer (5% PEG8000, 0.1% Triton X-100, 0.5 U/μL RNase inhibitor, 0.5 μM Smart-seq3 oligo-dT primer, and 0.5 mM dNTP) was incubated at 70°C for 2 min to denature the RNA. Next, reverse transcription mix (25 mM Tris-HCl pH 8.3­RT, 30 mM NaCl, 1 mM GTP, 2.5 mM MgCl2, 8 mM DTT, 0.5 U/μL RNase inhibitor, 2 μM TSO and 2 U/μL of maxima H-minus reverse transcriptase enzyme) was added to each sample. Reverse transcription and template switching were carried out at 42 °C for 90 min followed by 10 cycles of 50 °C for 2 min and 42 °C for 2 min. The reaction was terminated by incubating at 85 °C for 5 min. PCR pre-amplification was performed by directly adding KAPA HiFi HotStart ReadyMix and 0.5 μM forward primer and 0.1 μM reverse primer to a 50 μL reaction volume. The PCR was performed with the following cycle: 3 min at 98 °C for initial denaturation, 5–9 cycles (depending on the starting amount of RNA) of 20 s at 98 °C, 30 s at 65 °C, and 4 min at 72 °C. Final elongation was performed for 5 min at 72 °C. After PCR the cDNA was purified twice with 0.6x SPRIselect beads (Beckman Coulter, CA). Next, 600 pg of preamplified cDNA were tagmented with Tn5 transposase (Lucigen) pre-loaded with suitable adapters.  Libraries were further amplified for 14 cycles using custom Nextera-compatible primers with different indexes with KAPA HiFi HotStart ReadyMix. Libraries were loaded at 2.4 pM and sequenced on an Illumina Nextseq 500. The read configuration was 38 (read1), 8 (index1), 8 (index2), 38 (read2).