Solid 4 system

Genomic DNA was isolated from the tail using a DNeasy Blood and Tissue kit (Qiagen). Exome capture, the enrichment of exonic regions of genomic DNA, of SM/J and A/J were performed using a SureSelectXT Mouse All Exon kit (for AB SOLiD; Agilent Technologies) covering 49.6 MB (1.82%) of the mouse genome. We sequenced 50 base pairs of each tag in a single direction using a quarter of a cell of the SOLiD 4 system (Life Technologies) for each sample. Single nucleotide variants (SNVs) and indels (small insertions or deletions) were called by Avadis NGS with default parameters and detected 264,617 (SM/J) and 200,130 (A/J) SNVs/indels. SNVs and indels were compared to dbSNP Build 132. There were 623 exons in 63 genes between D2Mit380 at chr2: 69617675 (mm10) and D2Mit219 at chr2: 75416850 (mm10). The mean coverages of these exons were 32.9 for A/J strain and 24.9 for SM/J strain. The median coverages were 26 for A/J strain and 20 for SM/J strain. Nucleotide to nucleotide coverage counts revealed that 3.1% for A/J strain and 3.8% for SM/J strain were not covered by any reads. Exome data were deposited in DDBJ Sequence Read Archive (Accession No. DRA002145).

Our studies analyzed the RNA expression data collected by the studies done by Ramamoorthy et al. (2013 (link)). In those studies, “human hepatocytes from seven different subjects were obtained from CellzDirect (Durham, NC).” The primary human hepatocytes were grown on collagen coated plates and treated with rifampin (10 μM) or vehicle (methanol, 0.01%) for 24 h. Each culture from a different individual subject was treated as a biologic replicate (n = 7). In vitro studies were performed within 72–120 h from the time of hepatocyte isolation. Total RNA and miRNA were isolated with a miRNeasy kit (Qiagen, Valencia, CA). miRNAs (754) were analyzed using the Taqman OpenArray Human miRNA Panel in technical duplicates (each sample run on two OpenArrays). Global mRNA expression was measured by RNA-seq conducted on the SOLiD4 system (Life Technologies, Inc, Carlsbad, CA). The hepatocyte tissues were purchased commercially, and were de-identified. These studies were not deemed human subject research. CellzDirect was purchased by another company, and specific demographic or clinical information on the donors was not transferred in the transition and the information could not be retrieved.

Exome sequencing was performed on one affected child from all families using the SureSelect Human All Exon 50Mb Kit (Agilent Technologies UK, Cat. No G3370A). Sequencing was performed with the SOLiD4 System (Applied Biosystems) with 50 bp fragment reads (in families B and C) or the Illumina Analyzer IIx with 76 bp paired end reads (in families A and D). Raw sequencing reads were mapped to the GRCh37 reference human genome and changes compared to this reference sequence identified. Analyses focused on non-synonymous coding, nonsense, splice site variants, and indels involving exons. Potentially pathogenic mutations were identified based upon being unknown variants or those where the rare allele frequency was <1%, how well the site was conserved throughout evolution, and re-examination of the sequence reads containing potential mutations using the Integrated Genome Viewer. Analysis of the 84 genes in the candidate interval in family A revealed only one rare potentially pathogenic variant in PLAA (Table S2). This c.68G>T mutation in PLAA was found to segregate correctly by Sanger sequencing in families A, B, and C.

For each ChIP sequencing experiment, 50 μg chromatin and 4 μg antibody were used per IP. To obtain chromatin, brains from E12.5 embryos were harvested and the neocortical tissue was isolated in cold 0.5% glucose in PBS with 1× Protease inhibitor mixture (Sigma). The tissue was cross-linked immediately after harvesting with 1% formaldehyde (Thermo Scientific). Chromatin was sonicated using a Covaris S220 sonicator for 18 cycles of 60 s ON and 30 s OFF (5% Duty cycle, 2 Intensity and 200 cycles per burst) to get chromatin within the size range of 100–500 bp. The following antibodies were used for ChIP: goat α-LHX2 (Santa Cruz Biotechnology SC19344), goat IgG (Bangalore Genei). The protein-DNA complex was pulled down using Protein A-G magnetic beads (Dynabeads, Invitrogen). The immunoprecipitated DNA was purified using phenol-chloroform-isoamyl alcohol (Ambion). Sequencing libraries were prepared using SOLiD ChIP-Seq library preparation kit, and sequencing was performed on the SOLiD 4 System (Applied Biosystems). Five bases each were trimmed on the 5′ and 3′ ends, and reads were aligned to the reference genome mm⁹ using bowtie 1. Peaks were called using the MACS 1.4 program with default settings. The UCSC browser was used for data visualization.

Upon completion of PCR amplification, the small RNA libraries were purified using the SOLiD Library Micro Column Purification Kit (Applied Biosystems) and hybridized to the template beads using the SOLiD EZ bead system (Applied Biosystems). The template beads were amplified and deposited onto a tray for small RNA ligation sequencing by the SOLiD 4 System (Applied Biosystems). The sequencing data were uploaded to the Gene Expression Omnibus (GEO) with an accession number of GSE40049.

Faecal sample preparation and SOLiD (sequencing by oligonucleotide ligation and detection) shotgun sequencingwas performed as described previously [33 ]. Briefly, microbial DNA was isolated using the PSP Spin Stool DNA Plus Kit with lyses enhancer (Stratec Molecular, Berlin, Germany) as described by the manufacturer. Long-mate-paired libraries with mate-paired distances of 300–900 bp apart from the whole microbial DNA (with a maximum at 800 bp) were generated by randomly shearing in a microTube format using the Covaris S2 sonicator (Covaris, Woburn, MA, USA), according to the mate-paired library construction protocol (Applied Biosystems, Foster City, CA, USA). The fragmentation protocol was mildly adapted to duty cycle 5%, intensity 3, cycles per burst 200, at 4°C. Fragmentation times were adjusted to 15 sec. No size selection was performed. Finished libraries were clonally amplified on paramagnetic beads, deposited onto a glass slide, and sequenced according to standard Applied Biosystems protocols using the SOLiD 4 System (Applied Biosystems, Foster City, CA, USA).