GSM1438855
GSM1438855: Pcan42W; Polistes canadensis; Bisulfite-Seq
Genomic DNA and total RNA were extracted from single brains using the Qiagen All Prep DNA/RNA Mini kit according to the manufacturers’ instructions. For RNA-seq libraries, 50 to 200 ng total RNA was enriched for mRNA using Dynabeads Oligo(dT)25 from Invitrogen in two subsequent steps of purification with fresh beads. Fragmentation was done by incubation of mRNAs 5min at 94°C in first strand buffer (Invitrogen) and directly followed with cDNA synthesis using SuperScript III kit (Invitrogen). dUTPs were incorporated for second strand synthesis for libraries orientation. cDNA was end repaired, A-tailed and ligated with methylation Adaptor Oligo Kit (Ilumina) using the NEB Next kit according to the manufacturers’ instructions. dUTP excision was done prior to amplification using USER mix (NEB). Libraries were amplified with 16 cycles using 2x Phusion HF buffer (NEB). Size selection and cleaning between steps were performed with the AMPure XP system (Agencourt) to select DNA fragments between 250 and 500 bp. Paired-end libraries were sequenced on either Illumina GAIIx or HiSeq machines. For BS-seq libraries, synthetic genomes of P. canadensis and D. quadriceps were prepared using REPLI-g Ultra fast Mini kit from QIAGEN and 1 µL of amplification were treated in parallel with the other libraries. Libraries were prepared using 200 to 500 ng genomic DNA. After sonication (Covaris), DNA was end-repaired, A-tailed and ligated with methylation Adaptor Oligo Kit (Ilumina) using the NEB Next kit according to the manufacturers’ instructions. The adaptor-ligated DNA was treated with sodium-bisulfite using the Imprint DNA Modification Kit from Sigma-Aldrich according to the manufacturer’s instructions for the two-step protocol. Bisulfite-treated DNA was amplified using KAPA HiFi Uracil+DNA Polymerase with 15 cycles. Size selection and cleaning between steps were performed with the AMPure XP system (Agencourt) to select DNA fragments between 250 and 500 bp.
GEO
GSM1438855
GEO Accession
GSM1438855