Additional file 1 of T-RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells

Additional file 1: Figure S1. Schematic overview of the T-RHEX-RNAseq protocol outlining adapters, adapter introduction and primers used to amplify the library. In brief, double stranded cDNA reverse transcription with dUTP incorporated during the second strand synthesis is subjected to tagmentation with Tn5 loaded with i5 adapters. The i7 adapters are introduced by annealing an i7 oligo to the covalently attached part of the i5 adapter. Subsequently, gap fill in combination with ligation is used to covalently attach the i7 adapter. As Phusion is unable to utilize the dUTP containing strand as a template, stranded libraries are then generated by amplification using Pr2 in combination with the i5 completion primer. Figure S2. Strand-specificity of Tn-RNAseq and Directional Tn-RNAseq libraries. (A). Percentage of reads in exons: localized in a matched or mismatched orientation to transcript; or alternatively being localized in regions with overlapping antiparallel transcripts (undetermined). The data from Gertz et al., [1] was downloaded and processed using nf-core and strand-specificity evaluated using RSeq QC. Figure S3. Tracks and duplication rates of T-RHEX-RNAseq libraries from primary hematopoietic stem- and progenitor cells. (A) Tracks showing plus and minus strand reads in the Neat1, Kit, Hspd1 and Hspe1 genomic regions in primary mouse hematopoietic stem cells (HSCs) and lymphoid primed multipotent progenitors (LMPPs). Arrows below the gene names indicate the 5’-3’ direction of the transcript. RNAseq libraries were prepared directly from the indicated numbers of cells lysed in Single cell lysis solution (SCLS). The use of rRNA blocking reagents and dilution of the blocking reagent is indicated in parenthesis. (B) Reoccurrence (duplication rates) of reads in the indicated libraries. Figure S4. T-RHEX-RNAseq provides highly reproducible data. Spearman correlation between rlog of gene expression in samples generated from the indicated population. The use of rRNA blocking reagents and dilution of the blocking reagent is indicated in parenthesis. Hematopoietic stem cell (HSC with or without CD49b expression); Multipotent progenitor (MPP with no or low CD150 expression), lymphoid primed multipotent progenitors (LMPPs); granulocyte/monocyte progenitors (GMP); and antigen specific CD4 T cells (T, from wild-type or Bhlhe40 knockout mice). Data is from proof-of-principle experiments (HSC and LMPP; 250 and 500 cells respectively) or the subsequently generated T-RHEX-RNAseq data from antigen specific CD4 T cells (1000 cells) [1] and hematopoietic stem/progenitor cells (HSPCs; 250-500 cells) [2]. Table S1. Sample metrics and QC. Supplemental working protocol