title: GSM9209578: DSID18 Fetal membranes; human metagenome; OTHER
library_construction_protocol: All specimens were collected using sterile gloves, and all procedures were carried out under strict sterile conditions. For sequencing of 16S RNA genes and microbial community analysis, genomic DNA was extracted and the 16S rRNA amplified using V4 primers. 16S amplicon multiplex libraries were sequenced on Illumina platform at the Sequencing Facility of the Argonne National Laboratory. Genomic DNA was amplified using primers targeting the V4 region of the 16S rRNA gene with Illumina overhang adapters. Amplicon PCR was performed with KAPA HiFi HotStart ReadyMix under the following conditions: 95 °C for 3 min; 25 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; final extension at 72 °C for 5 min. PCR products were purified using AMPure XP beads (0.8× ratio). Dual indices and Illumina sequencing adapters were added in a second PCR (8 cycles) using Nextera XT index primers, followed by another AMPure XP bead cleanup. Libraries were quantified with Qubit and validated for size using Bioanalyzer. Equimolar pooling was performed prior to sequencing on an Illumina MiSeq platform (paired-end).