6000 nano labchip kit

Total RNA was extracted using TRIzol reagent (Invitrogen, CA, United States) following the manufacturer’s procedure. The 6000 Nano LabChip Kit (Agilent, CA, United States) with the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, United States) was used to assess RNA integrity. Approximately 1 μg of total RNA with an RNA integrity number (RIN) > 7.0 was used to prepare a small RNA library according to the protocol of a TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, United States). Then, we performed single-end sequencing (36 bp or 50 bp) on an Illumina HiSeq 2500. Approximately 10 μg of total RNA representing a specific adipose type was used to deplete ribosomal RNA with an Epicenter Ribo-Zero Gold Kit (Illumina). Following purification, the poly(A)- or poly(A)+ RNA fractions were fragmented into small pieces using divalent cations under elevated temperature. Then, the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for a mRNA-Seq sample preparation kit (Illumina), and the average insert size for the paired-end libraries was 300 bp (±50 bp). Then, we performed paired-end sequencing on an Illumina HiSeq 4000 (lc-bio, China) following the vendor’s recommended protocol.

The isolation of RNA was conducted in two independent biological replicates of non-infected BMDCs and three independent biological replicates of infected or fixed parasite-exposed BMDCs with the use of the Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) was used for RNA purification. Concentration and quality control of the isolated RNA were assessed using the ND-1000 Nanodrop microspectophotometer (ThermoFisher Scientific, Wilmington, DE, USA) and the 6000 Nano LabChip kit on the Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Palo Alto, CA, USA). Only RNA samples with RIN number > 7.5 were used for RNA Sequencing (RNA-Seq) using Ion Torrent technology.

Gastrocnemius muscles were homogenized in TRIzol (Invitrogen) and total RNA was extracted and purified using either RNeasy Mini or Micro kits (Qiagen) according to the manufacturer’s instructions. The quality of RNA was determined by electrophoresis using 6000 Nano LabChip kit on Bioanalyzer 2100 (Agilent). The samples without RNA degradation were used to synthesize cDNA using High Capacity Reverse Transcriptase kit (Applied Biosystems). Taqman ABI 7500 sequence detection system (Applied Biosystems) was used for real-time qPCR measurements on the samples using the delta-delta CT method [64 (link)]. qRT-PCR probes for Galgt1 and 18S RNA have been described in previous publications [32 (link),65 (link)]. Primers for dystrophin (Mm01216926_m1), Pax7 (Mm00834079_m1), Myf5 (Mm00435125_m1), MyoD (Mm00440387_m1), Myogenin (Mm00446194_m1), and Myh3 (Mm01332463_m1) were purchased from Applied Biosystems. 18S was used as an internal control. Measures shown are replicates of three to four muscles per condition.