Allprep dna extraction kit

DNA was isolated from stool samples using 0.25 g of stool thawed overnight at 4 °C. The repeated bead beating and column method was used [26 (link)], employing the TissueLyser II (Qiagen) with sterile zirconia beads (a mixture of 0.1 and 0.5 mm diameter). The stool samples were lysed by bead beating for 3 min at 30 Hz. To extract and purify bacterial DNA, the Qiagen AllPrep DNA extraction kit was used. DNA concentration for each sample was determined using a Nanodrop ND 1000 spectrophotometer.

Genomic DNA was isolated from maternal feces, placental tissue and maternal and infant buccal swabs. Fecal microbial DNA from 0.25 grams of thawed stool sample was extracted by repeated bead beating and column (RBB + C) method followed by Qiagen AllPrep DNA extraction kit as detailed in ref. 56 (link). DNA was isolated from buccal swabs and 100 grams of placental tissue by placing tissue in a 2 mL screw-cap tube containing 0.4 g of sterile zirconia beads (0.1 and 0.5 mm diameter) and 300 μL lysis buffer (NaCl 0.5 mol/L, Tris–HCl 50 mmol/L, pH 8.0, EDTA 50 mmol/L and SDS 4% w/v). Mechanical disruption was achieved by homogenization (Precellys, Bertin Technologies, Toulouse, France) for 3 min followed by incubation at 70 °C for 15 min. Oral and placental lysates were collected and transferred to the Maxwell 16 Buccal Swab LEV DNA Purification kit and to the Maxwell 16 Tissue DNA Purification kit (Promega, Madison, WI, USA) respectively, following the manufacturer’s recommendations. DNA extraction was carried out using the automated Maxwell 16 system. Contamination was monitored through assessment of reagent controls without addition of tissue or DNA. Extracted DNA was quantified by Nanodrop ND 1000 spectrophotometer (Nanodrop Technologies).

Total RNA was isolated from ~700K cells utilizing the AllPrep DNA extraction kit (Qiagen). ERCC RNA Spike-In Mix 1 was added to 100–250 ng of total RNA as outlined by the manufacturer (Ambion, Life Technologies). The ERCC control mix is a set of external RNA controls that enable performance assessment for gene expression experiments. The cDNA library was prepared with the TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Gold kit (Illumina). The concentration of each cDNA library was determined through qPCR (Kapa Biosystems). In all, 2 × 150 reads were generated on the HiSeq4000/NovaSeq6000 instrument (Illumina) generating ~83 million read pairs/sample.