title: GSM7162657: ISC_GFP-ss_biol rep 04; Drosophila melanogaster; RNA-Seq
library_construction_protocol: RNA from 5,000-30,000 cells was isolated using the Qiagen RNeasy Plus Micro Kit and eluted in 15µl water. RNA quality and concentration was analysed on a Bioanalyzer and only samples with RIN>7 were used for sequencing. NEBNext Low Input RNA libraries were prepared manually following the manufacturer's protocol (NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® Instruction Manual Version 5.0_5/20, NEB #E6420L). Samples were normalized to 1ng total RNA material per library in 8μl of nuclease-free water. RNA samples underwent reverse transcription, and the resulting cDNA was amplified by 10 cycles of PCR (according to the manufacturer recommendation for 1 ng input DNA). Amplified cDNA was subjected to two consecutive bead clean-ups with a 0.6X and 0.9X ratio of SPRISelect beads [B23318; Beckman Coulter] to sample volume. cDNA was fragmented enzymatically to target an insert size of ~200bp. Adaptors (diluted to 0.6µM) were ligated to the cDNA fragments and adaptor-ligated samples were cleaned up with SPRISelect beads (ratio: 0.8x). For the amplification of the sequencing library, 25µl of Q5 Master Mix was added, plus 10µl of a unique index (NEBNext Multiplex Oligos for Illumina [NEB #E6609]). Libraries were amplified by 8 PCR cycles. Final libraries were cleaned up with SPRISelect beads (ratio: 0.9x). The quality of the purified libraries was assessed using an Agilent D1000 ScreenTape Kit on an Agilent 4200 TapeStation.