Solid 4 system

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).

Raw sequences were generated using a SOLiD 4 System (Applied Biosystems). 50bp reads were output and demultiplexed according to barcode using SOLiD Instrument Control Software. The resulting sequence reads were aligned to the reference genome (hg19) using Bioscope Software (Applied Biosystems) with default settings. Data were expressed as reads per million mapped reads (RPM). RNAseq data described in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are available through GEO Series accession number GSE74102 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE74102).

The small RNA-seq was performed through the SOLiD platform. The PureLink miRNA isolation kit was used to construct the libraries of compatible fragments with SOLiD from an enriched fraction of small RNA. Sequencing microspheres were obtained by applying an emulsion PCR into an equimolar mixture of 48 libraries followed by an enrichment process before charging in the reaction chamber. Finally, the reaction to obtain the sequences (35 nucleotides + 10 nucleotides barcode) from the small RNA fraction was performed with the Applied Biosystems SOLiD 4 System. Samples were randomly distributed among the different sequencing slides to minimize batch effects. The data quality was estimated using the SOLiD Experimental Tracking System (SETS) software.

For the RNA sequencing of ligands bound to the HA-tagged proteins, RNA was extracted with the RNAeasy Mini Kit (QIAGEN) from three independent immunoprecipitation experiments. These were carried out with different batches of cytoplasmic extracts derived from wild-type L. infantum and transfected cells expressing HA-RBP23, HA-DRBD2 and the three HA-tagged PABPs. The RNA samples were quantified by Qubit RNA HS Assay Kit (Thermo Fisher) using to read the concentration the Qubit 2.0 Fluorometer. Between 0.1 to 4 μg of total RNA was used to construct the cDNA libraries with the TrueSeq Stranded mRNA Library Prep Kit (Illumina). The libraries were validated quantitatively through qPCR using the KAPA Library Quantification Kit and qualitatively by visualization in agarose gel. Finally, the libraries were normalized and pooled prior to sequencing using the MiSeq Reagent Kit v3, 150 cycle (Illumina). Similar RNA extractions were performed for the L. major IPs carried out with the native anti-PABP affinity purified antibodies, and total RNA was used to prepare cDNA libraries using the SOLiD Whole Transcriptome Analysis Kit followed by evaluation with an Agilent Bioanalyzer (Agilent). The cDNA libraries were used for clonal amplification according to the SOLiD Full-Scale Template Bead preparation protocol and sequenced with the SOLiD4 System (Applied Biosystems).