2200 tapestation instrument

Total RNA extracted with the use of Direct-zol RNA Mini Prep kit (Zymo Research) according to the protocol was further subjected to the quantity and quality controls using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and a TapeStation 2200 instrument (Agilent), respectively. NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) was used to prepare miRNA libraries. This protocol starts with the 3′ adaptor ligation, followed by hybridization with the Reverse Transcription Primer and ligation with the 5′ adaptor. Obtained products were reverse transcribed, and PCR amplified, including 12 different indexed primers to allow multiplexing of the samples. The libraries were then subjected to size-selection with Novex 6% TBE PAGE gel (Invitrogen) electrophoresis, followed by ethanol (POCH) purification and precipitation. The concentration of the obtained libraries was measured with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), while a 2200 TapeStation instrument (Agilent) was used to assess their size. The libraries mixed with the PhiX control library (Illumina) were clustered on an Illumina Flowcell_v3 in a cBot cluster station and then sequenced on HiScan SQ (Illumina) system according to the manufacturer protocol.

Tissues frozen in RNALater were thawed on ice in an RNAse-free work environment. Total RNA was extracted using a standard Trizol extraction protocol (Thermo Fisher Scientific, Waltham, MA), and RNA quality was assessed using the Tapestation 2200 Instrument (Agilent, Santa Clara, CA). Illumina sequence libraries were prepared using the TruSeq RNA Stranded LT Kit (Illumina, San Diego, CA), and library quality assessed using the Tapestation 2200 Instrument (Agilent, Santa Clara, CA). Each library was diluted to 2nM with sterile purified commercially available molecular biology-grade water (VWR) and pooled in a multiplexed library sample. The multiplexed library sample was then sent to the Novogene company for 125 base pair paired-end sequencing on a HiSeq 4000 platform.

Triplicates of each barcoded amplicon sample were combined. Each sample was diluted × 10⁵–10⁷-fold and the molar concentration of barcoded amplicons was quantified using a home-brew ddPCR library quantification assay and KAPA SYBR FAST Universal qPCR Library Quantification Kit (#KK4824, Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s instructions (the qPCR reaction was set up same as above).
Barcoded samples were pooled in equimolar amounts. The pooled library was purified using Agencourt AMPure XP beads (#A63880, Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions and eluted with ultrapure water (Invitrogen).
The purified library was confirmed to have the 260 nm to 280 nm light absorbance ratio of > 1.8 using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). The average amplicon size of approximately ~ 400 bp was confirmed with a High Sensitivity D1000 ScreenTape System (#5067-5584 and #5067-5585, Agilent Technologies, Santa Clara, CA, USA) using a 2200 TapeStation instrument (Agilent Technologies) and the Agilent 2200 TapeStation Software A.02.01 (Agilent Technologies).
The molar concentration of the pooled library was measured using the ddPCR and KAPA qPCR assays, and the library was submitted for NGS with the sequencing primers described in Additional file 1: Table S1.

Total RNA was extracted from 68 samples of pre- and post-NAC tumor tissues using RNeasy Plus Mini Kit DNase I digestion (Qiagen, Germany #74134), and RiboLock RNase inhibitor (Fermentas, Lithuania #EO0382) was added to the isolated RNA. Five patients showed a complete response, rendering it impossible to obtain further tumor samples. The RNA integrity number (RIN) was measured using the 2200 TapeStation Instrument and R6K ScreenTape (Agilent Technologies, USA #5067–5367). RNA with an RIN > 7 was reverse transcribed to cDNA using the RevertAid Kit with random hexanucleotide primers (Fermentas, Thermo Scientific #K1691) following the manufacturer's instructions.

DNA was extracted from 68 samples of pre-NAC tumor tissues using the QIAamp DNA mini Kit (Qiagen, Germany #51304), and the DNA concentration and purity were assessed using a NanoDrop-2000 spectrophotometer (Thermo Scientific, USA). The concentration level ranged from 50 to 150 ng/μl; the A₂₆₀/A₂₈₀ = 2.10–2.35; and the A₂₆₀/A₂₃₀ = 2.15–2.40. The integrity was evaluated by capillary electrophoresis using the 2200 TapeStation Instrument and Agilent Genomic DNA ScreenTape System Quick Guide (Agilent Technologies, USA # 5067–5365). The DNA mass was greater than 48 kbp.

Primers were designed with NCBI Primer-BLAST tool with Tm range of 59–61 °C. The universal primer sequences (CS1 and CS2) were added at the 5′ end of the designed primers. All primer pairs were tested alone and in multiplexed PCR reactions using 10 ng of TaqMan® Control Human Genomic DNA (Thermo Fisher Scientific) in 10 μl reaction volumes. The coverage and performance of primers were analysed using 2200 TapeStation instrument (Agilent) and Hi-seq 4000. The primers were grouped together as 7-8plex, and primers in each group were chosen to target different genes in order to minimise non-specific amplification and cross-reactivity.