Nanodrop eight

CT26 tumor tissues were isolated and total RNA was extracted using the RNeasy 96 QIAcube HT Kit (Qiagen; C/N 74171) according to manufactuers guidelines with a DNase digest included. RNA concentration was determined by Qubit Flex Fluorometer (Invitrogen), RNA purity was determined using a NanoDrop Eight (Thermo Scientific) and RNA integrity was measured using a 4200 Tapestation (Agilent). All samples had a RINe of ≥7.0. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs; #E7760L) as per manufactuer’s guidelines with 1000 ng of RNA input. Ribosomal RNA was removed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs; E7490L). Libraries were subjected to 9 cycles of PCR with unique dual indexes (New England BioLabs; E6440L). Libraries were subsequently quantified by Qubit Flex Fluorometer (Invitrogen), and library sizes were determined by 4200 Tapestation (Agilent) and pooled equimolar. Each library was loaded onto one lane of an S4 v1.5 flow cell (300 cycles) (Illumina, #20028312) on an Illumina NovaSeq 6000 at 2 × 150 paired-end configuration.

The genomic DNA from cotton plant leaf tissues was isolated using the cetyltrimethylammonium bromide (CTAB) method [51 (link)]. The DNA concentration was calculated by measuring the absorbance of 1 µL of the samples at 260/280 nm using the NanoDrop Eight spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA samples were diluted to a working concentration of 25 ng/µL. A total of 72 SSR (simple sequence repeat) markers were selected from CottonGen the cotton marker database (https://www.cottongen.org/data/markers) (accessed on 20 February 2022) [52 (link)]. PCR-based SSR genotyping was conducted as described previously [6 (link),53 (link),54 (link)]. The construction and visualization of the phylogenetic tree was performed using NCSS 12.