GSM8767678: Plate 1, biological replicate 2, tagmentation technical replicate 1; Homo sapiens; RNA-Seq
Cells were lysed in TRK lysis buffer (Omega Bio-tek) and incubated with Proteinase K. RNA was isolated using 30% PEG beads prepared in house (Thermo Sera-Mag Speed Beads #45152105050250 resuspended in 2M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.01% Igepal CA-630, 0.05% sodium azide, 30% PEG 8000) at a 1:2 ratio of lysate to beads. Samples were treated with DNase I (Thermo) in the presence of RNasin Plus Ribonuclease Inhibitor (Promega). Prime-seq (https://www.protocols.io/view/prime-seq-81wgb1pw3vpk) was adapted for library preparation. During cDNA synthesis, prime-seq uses poly(A) priming, template switching, early barcoding, and UMIs to generate 3′ tagged RNA-seq libraries. Following reverse transcription into cDNA, samples from all wells of a 96-well plate were pooled with 30% PEG beads at a ratio of 1:1 lysate to beads. Pooled samples were then treated with Exonuclease I (Thermo). cDNA was then amplified using Terra PCR Direct Polymerase Mix (Takara) for 9 cycles. Libraries were then tagmented (4-5 technical replicates per library) and amplified using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer's instructions.
GEO
GSM8767678_r1