Trueseq library preparation kit

Selective chemical labeling-exonuclease (SCL-exo, (35 (link))) experiments were conducted on starved cells after 50 min of E2 (10 nM)/ethanol treatment. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (69506; QIAGEN). For each experiment, 8 μg of gDNA was sonicated two times for 7 min (30 s on/30 s off) and two times for 14 min (30 s on/30 s off) with a Bioruptor (Diagenode). Glucosylation and biotinylation of 5hmC were performed with the Hydroxymethyl Collector kit (55013; Active Motif), followed by on beads-exonuclease digestion of the captured fragments and library preparation (TrueSeq library preparation kit, IP-202-1012; Illumina). For normalization purpose, 400 pg of 5hmC control DNA provided by the Hydroxymethyl Collector kit was added to each sample as spike-in. Libraries from seven independent SCL-exo experiments were sequenced on seven lanes of a HiSeq 1500 (Illumina) by the GEH facility.

Corneas were removed from the eyes by excision. Corneal tissue was immediately homogenized in Trizol reagent (Invitrogen) and the total RNA was isolated using Direct-zol RNA Miniprep kit according to the recommended protocol (Zymo Research). To eliminate potential contamination with genomic DNA (gDNA) the samples were subjected to additional treatment with recombinant DNase I (Ambion) for 30 min at 37°C and the total RNA was re-purified over RNeasy mini columns (Qiagen). Quality and the RNA concentration in the samples were evaluated using the RNA chip bioanalyzer (Agilent Technologies). As an additional step to test RNA quality, we performed RT-PCR assays using a panel of primers for several ubiquitously expressed genes including Gapdh and G protein subunits (Gnb1, Gnai2). Samples were depleted of both cytoplasmic and mitochondrial ribosomal RNA using Ribo-Zero-Gold kit (Epicentre). Library preparation for next generation sequencing was performed using Illumina TrueSeq library preparation kit that allows for paired-end directional RNA sequencing. Next-generation sequencing of corneal RNA was performed at the Hussman Institute for Human Genomics core facility using the Illumina HiSeq 2000 platform.

Nuclei were incubated in 50 uL of Tn5 transposition buffer for 30 min at 37°C. Library preparation with Illumina TrueSeq library preparation kit and paired-end Illumina HiSeq 2500 sequencing (80 million reads per sample) was performed at the University of California, Irvine High Throughput Genomics Facility. Motif analysis was conducted with MEME Suit 5.5.0 using predicted IRX5 motif (JASPAR, 2020 #PH0086.1).

Library preparation was done with Illumina TrueSeq library preparation kit, and single-end Illumina HiSeq 2500 sequencing was performed.

cDNA libraries were prepared with 500 ng of total RNA using the TrueSeq® Library preparation kit (Illumina, USA). Quality control for libraries was done using the Bioanalyzer DNA 1000 (Agilent Technologies, USA), and dsDNA concentration was measured with Qubit 2.0 Fluorometer (Life Technologies, USA). The HiSeq 2000 sequencing platform (Illumina, USA) was used to perform 50 bp single‐end sequencing on the samples.

For RNA-seq from uterine tissues of implantation sites, total RNA was isolated by TRIzol extraction as described above. RNA concentration was determined via a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA). RNA integrity analysis, library preparation, and sequencing were performed by the Yale Center for Genomic Analysis. RNA integrity was measured using an Agilent 2000 Bioanalyzer utilizing the Agilent RNA 6000 Pico Chip (Agilent, Santa Clara, CA) per the manufacturer’s specifications. Library preparation was performed with the Illumina TrueSeq Library Preparation Kit (Illumina, San Diego, CA) per the manufacturer’s specifications. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP for generating strand-specific sequencing libraries. Libraries were sequenced on an Illumina HiSeq2500 with parameters set for high output, single-end chemistry, and 76-bp sequencing. Samples were multiplexed to six samples per lane, yielding an average of 37 million reads per sample.

Total RNA was isolated from EVs by Qiagen RNeasy Plus Micro Kit, and RNA concentration was determined by NanoDrop 2000 (Thermo Fisher Scientific). RNA quality analysis, library preparation, and sequencing were performed by the Yale Center for Genomic Analysis. RNA was measured using an Agilent 2000 Bioanalyzer, which utilized the Agilent RNA 6000 Pico Chip (Agilent, Santa Clara, CA) per the manufacturer’s specifications. Illumina TrueSeq Library Preparation Kit (Illumina, San Diego, CA) was used for RNA-seq library preparation in accordance with the manufacturer’s protocol. Random primers were used for first-strand synthesis and then second-strand synthesis was performed with dUTP for generating strand-specific sequencing libraries. The parameters were set for single-end chemistry, high output, and 56-bp sequencing. Samples were multiplexed to six samples per lane. Libraries were sequenced on an Illumina HiSeq2500.