Hiseq 2500 apparatus

Prior to RNA isolation, tissue was disrupted in the homogenization buffer of the RNA extraction protocol using a Tissue Lyser instrument (Qiagen). RNA was isolated using the RNeasy Mini kit (Qiagen), performing specialized protocols for brain and muscle tissues as recommended by the manufacturer. The RNA was treated with DNase I on the affinity column before elution. Strand specific RNA-seq libraries, including poly-A(+) mRNA selection and RNA fragmentation, were prepared using the TruSeq Stranded RNA LT Kit (Illumina) according to the supplier’s instructions, with 2 μg total RNA as input. The resulting libraries had insert sizes of ca. 100–400 bp as indicated by DNA 7500 Chips run on an Agilent Bioanalyzer 2100 instrument (Agilent). All ten libraries were combined into a single pool. Sequencing of 200-nt paired-end reads was performed using an Illumina HiSeq 2500 apparatus in Rapid mode with TruSeq Rapid SBS chemistry on two lanes (Illumina). Read data for each library were extracted in FastQ format using the CASAVA software v1.8.4 (Illumina) using default settings.

For RNA-seq, total RNA from WT PC, KO PC and GB, single and co-cultured PC-GB, was extracted with the purification RNA RNeasy Mini Kit following manufacturer instructions and treated with DNase I (Qiagen). Equal amounts of purified total RNA from three to four experiments of each one were pooled in each sample. DNA libraries for small RNAs and mRNAs were processed and sequenced by the CRG core genomics facility (Barcelona, Spain) using a HiSeq-2500 apparatus (Illumina, service provided by Fasteris S.L.) according to the manufacturer’s instructions. For the quality control, read alignment, obtaining metrics for gene expression, please seeSupplementary Text. Differentially expressed genes (DEGs) between GB conditioned PC (GB-WT PC) and CMA-deficient PC with GB (GB-KO PC) were detected using DESeq2 v1.18.1 package (Love et al., 2014 (link)) in R computing platform v3.4.4 (Ihaka and Gentleman, 1996 (link)). DEGs were computed using batch correction in the formula design (design = ∼condition + sample_batch). Genes with FDR Adj. p < 0.01 were considered significantly differentially expressed. Raw data are publicly available in the European Nucleotide Archive ENA (study ID PRJEB48545).

RUES2 cells were seeded in 6-well plates (200,000 single cells per well) in E7 medium supplemented with 10 µM Rock-inhibitor and incubated overnight. On the following day the media was changed to E7 supplemented with 10 µM Rock-inhibitor with or without 10 ng/mL ACTIVIN A. Samples (three pooled-wells) were collected in 1 mL Trizol at 1, 2.5, 4, 8 and 12 hr for the ACTIVIN-treated conditions and after 0, 6 and 12 hr for the no-ACTIVIN conditions (to be used as negative controls). Total cellular RNA for each sample was extracted using RNeasy Mini Kit (QIAGEN) and 2 ug of total RNA was used to prepare each individual RNA-seq library. RNA-seq library construction was conducted with the TruSeq RNA Library Preparation Kit (Illumina, San Diego, CA) as per the manufacturer’s instructions and sequenced in an Illumina HiSeq 2500 apparatus. Raw reads were mapped to hg19 using STAR aligner, and the gene read counts were normalized using the DESeq2 Bioconductor package (Love et al., 2014 (link)). Library preparation, sequencing, and mapping were performed by the New York Genome Center (New York, NY, USA). All raw data files are available from the GEO database (accession number GSE111717).