title: GSM3539246: DM014: Patient29_Plate1; Homo sapiens; RNA-Seq
library_construction_protocol: Single CD3+T cells from patient BAL were sorted directly into wells of 96-well plates; each well contained 7.5uL lysis buffer: 0.2% Triton X-100 (Sigma), 1 U/uL SUPERaseIN (ThermoFisher), 2mM dNTPs (ThermoFisher), 2uM RT primer (Integrated DNA Technologies). Primer annealing was performed at 72°C for 3 minutes. Reverse transcription was performed by adding 7.5uL RT mix to each well (2M Betaine (Affymetrix), 2X Protoscript Buffer (New England Biolabs), 12mM MgCl2 (ThermoFisher), 10mM DTT (ThermoFisher), 5.3U Protoscript II Reverse Transcriptase (New England Biolabs), 0.53U SUPERaseIN (ThermoFisher), 2uM Template Switching Oligo (Integrated DNA Technologies, table S6). Reverse transcription was performed at 42°C for 90 minutes, followed by 10 cycles of 50°C for 2 minutes, 42°C for 2 minutes, 70°C for 10 minutes, followed by a 4°C hold. Excess primers were removed by adding 2ul Exonuclease I (ThermoFisher) mix to each well (1.875U ExoI in water) and incubating at 37°C for 30 minutes, 85°C for 15 minutes, 75°C for 30 seconds, 4°C hold. All wells were pooled into a single 15-ml falcon tubes and cDNA was purified and concentrated using Dynabeads™ MyOne™ Silane beads (ThermoFisher) according to the manufacturer's instructions. The cDNA was split into duplicate reactions containing 25ul cDNA, 25ul 2x HIFI HotStart Ready Mix (Kapa Biosystems), and 0.2M SMART PCR Primer (table S6), PCR amplified as above, and duplicate reactions were combined and purified using 0.7 volumes AMPure XP beads (Beckman Coulter). The amplified cDNA was visualized on an Agilent TapeStation and quantified using a Qubit II fluorometer (ThermoFisher). Sequencing libraries were constructed using Nextera XT (Illumina) with modifications. A custom i5 primer was used (NexteraPCR, table S6) with 0.6ng input cDNA and 10 cycles of amplification was performed. Unique i7 indexes were used for each plate. After amplification, the library was purified with two rounds of AMPure XP beads, visualized on the TapeStation and quantified using the Qubit II fluorometer. Libraries were sequenced on an Illumina NextSeq 500 using the 75 cycle High Output kit (read lengths 26(R1) x 8(i) x 58(R2)). Custom sequencing primers were used for Read 1 (SMART_R1seq and ILMN_R1seq, see table S6). With each plate we targeted ~70M reads. Library pools were loaded at 1.8pM with 30% PhiX (Illumina). Pooled, 3'-end single-cell RNA-seq
identifiers.namespace: GEO
identifiers.id: GSM3539246