GSM9141054: Control replicate 3; Drosophila melanogaster; RNA-Seq
For sample preparation for RNA sequencing, embryos were manually sorted to avoid non developing embryos and to select for only cellularizing and gastrulating embryos. Firstly, egg laying was synchronised by discarding the first 2hr collection. For genotypes which laid enough embryos, 1hr collection was taken and aged for 1.5-2 hrs whereas for mutants which did not lay enough embryos 2.5-3 hr collection was taken directly after synchronisation from cages at 28 C. Embryos were dechorionated using bleach and washed with distilled water. Then around 50-60 embryos were taken, using a brush, on a slide and around 20-30ul 1X PBS was added on top of the embryos. Embryos of desired stages were then sorted using a single haired brush under a stereo microscope. These embryos were taken in an MCT and crushed in TriZol (Qiagen) using a homogeniser gun, snap frozen in liquid nitrogen and then stored at -80C. Multiple embryo collections were pooled such that there were 70-100 embryos (equal proportion of cellularizing and gastrulating embryos) per each replicate. RNA was isolated using the TRIZOL method. The concentration of RNA was measured using Qubit RNA BR Assay (Invitrogen, Cat# Q10211). RNA purity was checked using QIAxpert and RNA integrity was checked on TapeStation using RNA ScreenTapes (Agilent, Cat# 50675579). KAPA RNA HyperPrep Kit (cat #0000141759) protocol was used to prepare libraries for mRNA sequencing. An initial Concentration of 200ng of total RNA was taken for the assay. mRNA molecules are captured using magnetic oligo Poly(T) beads (KAPA mRNA capture kit, Cat# 7962240001). Following purification, the enriched mRNA were fragmented using divalent cations under elevated temperatures (94 degrees for 6 minutes). The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random priming. Combined 2nd strand synthesis and A-tailing is done, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), incorporates dUTP into the second cDNA strand, and adds dAMP to the 3' ends of the resulting dscDNA. This is followed by Adapter Ligation, in which dsDNA indexing adapters with 3' dTMP overhangs are ligated to library insert fragments. The adapter-ligated products were then purified and enriched carrying appropriate adapter sequences at both ends using high-fidelity, low-bias PCR ( The strand marked with dUTP is not amplified, allowing strand-specific sequencing) using the following thermal conditions: initial denaturation 98°C for 45sec; 13 cycles of - 98°C for 15sec, 60°C for 30sec, 72°C for 30sec; final extension of 72°C for 1mins. PCR products are then purified and checked for fragment size distribution on Fragment Analyzer using HS NGS Fragment Kit (1-6000bp) (Agilent, Cat# DNF-474-1000). Prepared libraries were quantified using Qubit HS Assay (Invitrogen, Cat# Q32854). The obtained libraries were pooled and diluted to final optimal loading concentration. The pooled libraries were then loaded on to Illumina NovaSeq V1.5 instrument to generate 40M, 150bp paired-end reads/sample.