Second strand synthesis module

Total RNA was isolated using TRIzol LS/chloroform extraction of total RNA, followed by EtOH precipitation and column-based cleanup using Direct-zol tubes (Zymo Research). Ribosomal RNA was reduced in each sample using Ribo-Zero (Illumina) and purified using SPRI beads (Agencourt RNAClean XP). Immediately following the RNA cleanup, first-strand cDNA synthesis was performed using SuperScript IV (Invitrogen), followed by second-strand synthesis using the second-strand synthesis module (NEB). Shearing of cDNA was performed using a Covaris LE220 and end repaired using the NEB end repair module. Y-Adapter ligation to A-tailed cDNA was followed by QPCR-based barcoding of samples.

BRB-seq [9 (link)] was performed for library preparation with the following modifications. Barcoded oligo-dT-based primer (5′-GCCGGTAATACGACTCACTATAGGGAGTTCTACAGTCCGACGATCNNNNNNNNNNCCCCCCCCCTTTTTTTTTTTTTTTTTTTTTTTTV -3′; 10bp “N” = UMI, 9bp “C” = cell barcode) was used for single-stranded synthesis and Second Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA, #E6111) was used for double-stranded cDNA synthesis. In-house MEDS-B Tn5 transposase [27 (link),28 (link)] was used for tagmentation, and libraries were amplified for 10 cycles of PCR using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA, #M0530) with the following primers (5′ -AATGATACGGCGACCACCGAGATCTACACindexGTTCAGAGTTCTACAGTCCGA-3′,5′-CAAGCAGAAGACGGCATACGAGATindexGTCTCGTGGGCTCGGAGATGT-3′). An Illumina NovaSeq6000 (Illumina, San Diego, CA, USA) was used to obtain 15 bp of barcode read (Read1) and 81 bp of insert read (Read2).

RNA was thawed on ice before being normalized to 50 ng/μL in order to obtain uniform library sizes. Bulk gene expression with RNA sequencing was performed on whole blood samples from the two intervention groups and placebo at baseline and endline to assess the molecular effect of zinc supplementation on peripheral blood gene expression, and to probe for a possible biomarker for zinc supplementation status. Transcriptome libraries were generated by adapting the CelsSeq2 protocol [12 (link)] as follows: Samples were pooled after first-strand cDNA synthesis and treated with Exonuclease 1 for 30 min, followed by a 1.2X bead clean-up. Second-strand synthesis was performed using the NEBNext Second Strand Synthesis module (NEB #E6111S) in a final reaction volume of 20 μL, and NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel—7449970.5) were used for all DNA purification and size selection steps.