2100 bioanalyzer high sensitivity dna analysis kit

NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina and NEBNext rRNA Depletion Kit (NEB, Ipswich, MA) was used to generate RNA-seq libraries from purified RNA following the manufacturer’s instructions. Libraries were quantified with the Qubit High Sensitivity dsDNA Assay (Invitrogen, Carlsbad, CA), and size distribution was assessed with a 2100 Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent). Multiplexed bulk human placental libraries were sequenced on the NextSeq500 platform using 150 cycle single-end protocol generating a total of 36.9 to 70.9 million 101bp reads per sample. Multiplexed rhesus trophoblast cell lines were sequenced on the NextSeq500 platform using 100 cycle single-end protocol generating a total of 57.8 to 68.5 million 75bp reads per sample.

Four to 15 samples were pooled to each capture reaction. Equal nanograms of each library were in each pool (13–40 ng per sample). The capture was performed according to SeqCap EZ Library SR User’s Guide v5.0 protocol with the following exceptions using SeqCap EZ Choice Library (Roche Sequencing, Pleasanton, CA, USA). For the test samples HE oligos were used and for the dual index libraries 1 µl per xGen Universal Blocking oligo Truseq HT-i7 and i5 (Integrated Device Technology, San Jose, CA, USA) were used instead of the HE oligos. Ten cycles were used for post-capture amplification instead of 14. The captured and amplified pools were quantified with 2100 Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent Technologies, Santa Clara, CA, USA).