GSM4251911: Input_3; Homo sapiens; RNA-Seq
Recovered cells were centrifuged at 600g for 5 min and supernatant was replaced with 25 µl of PBS containing 0.01 % bovine serum albumin (Sigma). Subsequently 25 ml of lysis buffer were added, containing 0.1 % Sarkosyl (Sigma), 5 mM EDTA (Life Technologies), 0.1 M Tris (pH 7.5, Sigma) and 25 mM 1,4-Dithioreitol (DTT, Sigma), 800 units/ml RNase inhibitor (NEB) and 500 Drop-Seq beads (Beads, Lot 120817, ChemGenes). All beads pelleting steps were carried out at 1000 g for 1 min in 1.5 ml microtubes (Axygen). Reverse transcription (RT), exonuclease I (ExoI) treatment and PCR were performed as described Macosko et al. with minor adaptations30. Lysed cells were incubated at 1400 rpm for 5 min at room temperature and subsequently washed twice with 1 ml of 6x SSC buffer (Sigma). Reverse transcription was performed for 90 min at 42°C in 50 µl of 1 mM dNTPs (Clontech), 2.5 µM template switch oligo (see Table 1), 1250 units/ml RNase inhibitor, 1x Maxima RT buffer and 10,000 units/ml Maxima H minus reverse transcriptase (Thermo Fisher Scientific). Drop-seq beads were washed twice with 0.5 % SDS (Applichem) in 10 mM Tris (TE‑SDS), twice with 0.01 % Tween‑20 (Sigma) in 10 mM Tris (TE‑TW) and once with 10 mM Tris pH 7.5. The Drop-seq beads pellet was then incubated with 50 µl of exonuclease mix containing 1x Exonuclease I Buffer and 1000 units/ml Exonuclease I (NEB), and incubated at 37°C for 45 min at 1,400 rpm). Drop-seq beads were washed twice with TE‑SDS, twice with TE‑TW and once with double-distilled H2O. Beads were amplified by PCR in 25 µL of 1x Hifi HotStart Readymix (Kapa Biosystems) and 0.8 µM TSO-PCR primer (Table 1) at 95 C 3 min; 4 cycles of: 98 C for 20 sec, 65 C for 45 sec, 72 C for 3 min; then 16 cycles of: 98°C for 20 sec, 67°C for 20 sec, 72°C for 3 min and an extension step of 5 min. Libraries were purified using Ampure XP beads (0.6x ratio to remove small fragments), cDNA was quantified using a Qubit HS kit (Thermo Fisher Scientific) and integrity analyzed on a Fragment Analyzer (Agilent). Libraries were prepared using in-house produced Tn5 loaded with adapters, as described31. Size selected and purified libraries were sequenced paired-end on a NextSeq 500 system (Illumina) in High-Output mode following recommendations from the original protocol (read 1 20 bp and read 2 50 bp)
GEO Accession
GSM4251911