Sitophilus oryzae

RNAseq of midgut of day 5 Sitophilus oryzae, replicate 2

Total RNA was extracted using the Qiagen Allprep DNA/RNA micro kit (Qiagen 80284) following the manufacturers protocol. RNA quality was assessed by the national platform GenomEast, along with library construction and sequencing. RNA-Seq libraries were generated according to manufacturer's instructions from 200 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94oC for 2 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.

{'age': 'Day 5 adult', 'breed': 'Bouriz', 'tissue': 'Midgut', 'geo_loc_name': 'France', 'biosamplemodel': 'Invertebrate', 'collection_date': '2011-12-01', 'isolation_source': 'rice, France', 'host_tissue_sampled': 'midgut'}

RNAseq of midgut of day 5 Sitophilus oryzae, replicate 3

Total RNA was extracted using the Qiagen Allprep DNA/RNA micro kit (Qiagen 80284) following the manufacturers protocol. RNA quality was assessed by the national platform GenomEast, along with library construction and sequencing. RNA-Seq libraries were generated according to manufacturer's instructions from 200 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94oC for 2 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.

{'age': 'Day 5 adult', 'breed': 'Bouriz', 'tissue': 'Midgut', 'geo_loc_name': 'France', 'biosamplemodel': 'Invertebrate', 'collection_date': '2011-12-01', 'isolation_source': 'rice, France', 'host_tissue_sampled': 'midgut'}

RNAseq of midgut of day 5 Sitophilus oryzae, replicate 4

Total RNA was extracted using the Qiagen Allprep DNA/RNA micro kit (Qiagen 80284) following the manufacturers protocol. RNA quality was assessed by the national platform GenomEast, along with library construction and sequencing. RNA-Seq libraries were generated according to manufacturer's instructions from 200 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94oC for 2 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.

{'age': 'Day 5 adult', 'breed': 'Bouriz', 'tissue': 'Midgut', 'geo_loc_name': 'France', 'biosamplemodel': 'Invertebrate', 'collection_date': '2011-12-01', 'isolation_source': 'rice, France', 'host_tissue_sampled': 'midgut'}