AR pro, Library improvement
Homo sapiens
Protocols: To synthesize first-strand cDNA, we added 10 µL of reverse transcription (RT) reaction mix containing 1× RT Buffer (EP0751, Thermo Scientific), 1 mM dNTP mix, 2 U/μL Recombinant RNase inhibitor, 4 μM biotinylated template-switching oligo, and 4 U/μL Maxima H Minus Reverse Transcriptase (EP0751, Thermo Scientific) to 10 µL of the hydrogel bead suspensions. We incubated the reaction mixture at 42˚C overnight and then heat-inactivated the enzyme at 85˚C for 5 min.  We rescued cDNA fragments that failed template switching by performing second-strand synthesis. Specifically, we incubated the beads in 100 µL of 0.1 N NaOH for 5 min, washed them three times with 100 µL of TE–TW (5 mM Tris–HCl pH 8.0, 0.1 mM EDTA, 0.01% Tween-20), and finally resuspended them in 10 µL of TE–TW. We synthesized second-strand cDNA in a buffer containing 1 mM dNTP, 10 µM dN-SMART primer, and 0.13 U/µL Large (Klenow) Fragment (New England Biolabs) by incubating the reaction at 37℃ for 60 min followed by 75℃ for 20 min. We then washed the beads three times with 100 µL of TE–TW and once with 50 µL of nuclease-free water, yielding a final sample volume of 20 µL. We amplified the synthesized DNA in 50 μL PCR reaction containing 0.24 μM primer2 (AAGCAGTGGTATCAACGCAGAGT), 9 nM additive primer (AGACGTGTGCTCTTCCGATCT), 1× SeqAmp PCR Buffer, and 0.025 U/μL SeqAmp DNA Polymerase (Takara) under the following cycling conditions: 95℃ for 1 min; 15–17 cycles of 98℃ for 10 s and 65℃ for 30 s; 68℃ for 4 min; and 72℃ for 10 min. We size-selected long cDNA fragments derived from mRNA using SPRIselect beads (B23318, Beckman Coulter) at a 0.6× followed by 0.8× bead-to-sample volume ratios. We purified the first supernatant, which containing the short DNA tag library (<200 bp), using a 2× bead-to-sample ratio. We eluted purified DNA in 13 or 11 μL of elution buffer (10 mM Tris-HCl, pH 8.5). We quantified the long cDNA yield and quality using a Qubit 4 Fluorometer (Thermo Fisher Scientific) and quantitative real-time PCR (qPCR) targeting GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Hs02758991_g1, Thermo Fisher Scientific). We performed subsequent procedures for library preparation as previously reported (Tsuchida et al., Lab on a Chip, 2024). Briefly, we fragmented long cDNA and carried out PCR using Nextera XT (Illumina) with custom index primers, followed by purification using AMPure beads at 0.6× the reaction volume. We quantified the DNA tag library using KAPA Library Quantification Kits (Roche) and sequenced the library on an Illumina NextSeq 550 with paired-end reads.
isolate
Mixture of mRNA from the HeLa cells
age
not applicable
biomaterial_provider
RIKEN BRC
collection_date
not applicable
geo_loc_name
not applicable
sex
female
tissue
cervix
cell_line
HeLa
cell_type
epithelial cell
disease
cervical carcinoma
sample_type
RNA
BioSampleModel
Human