TITLE: SmartSeq2 for HTP Generation of Bulk RNA Libraries- with Pipetting plan 
AUTHORS: Sasa T, Matjaž H

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">[insert abstract from MACA_Bulk paper]</div></div>

[STEPS]
?. [cDNA Synthesis]
First Strand Synthesis cDNA synthesis was performed using the Smart-seq2 protocol1. Briefly, 96-well plates containing bulk RNA were thawed on ice followed by first-strand synthesis. 6 μl of RT Master Mix was added to each well and mixed by hand. Reverse transcription was carried out by incubating wells on a Biorad S1000 thermal-cycler (BioRad) at 42 °C for 90 min, and stopped by heating at 70 °C for 5 min. Pipetting plan: RT Master Mix16.7 U/μl SMARTScribe Reverse Transcriptase (Takara Bio, 639538)1.67 U/μl Recombinant RNase Inhibitor (Takara Bio, 2313B)1.67x First-Strand Buffer (Takara Bio, 639538),1.67 μM TSO (Exiqon, 5′-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3′)8.33 mM dithiothreitol (Bioworld, 40420001-1)1.67 M Betaine (Sigma, B0300-5VL)10 mM MgCl2 (Sigma, M1028-10X1ML)1. Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10, nmeth.2639 (2013).
?. [cDNA Synthesis]
2nd Strand Synthesis and cDNA AmplificationSubsequently, 15 μl of PCR mix was added to each well and mixed by hand, and second- strand synthesis was performed on a Biorad S1000 thermal-cycler by using the following program: cDNA PCR Master Mix1.67X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602)0.17 μM IS PCR primer (IDT, 5′-AAGCAGTGGTATCAACGCAGAGT-3′)0.038 U/μl Lambda Exonuclease (NEB, M0262L))PCR Conditions: 1) 37 °C for 30 min2) 95 °C for 3 min3) 6 cycles :    98°C for 20s    67°C for 15s    72°C for 4min4)72°C for 5min.
?. [Library Pooling, QC, and Sequencing]
Pooled libraries were split and sequenced on the NovaSeq 6000 Sequencing System (Illumina) using 2 × 100-bp paired-end reads and 2 × 12-bp index reads with 2 x S4 300-cycle kit (Illumina, 20012861 or 20012860).
?. [Library Pooling, QC, and Sequencing]
After library preparation, wells of each library plate were pooled using a Mosquito liquid handler. Pooling was followed by two purifications using 0.7x AMPure beads (Fisher, A63881). Library quality was assessed using capillary electrophoresis on a Fragment Analyzer (AATI) or Tapestation (Agilent), and libraries were quantified by qPCR (Kapa Biosystems, KK4923) on a CFX96 Touch Real-Time PCR Detection System (Biorad). Pooled libraries were normalized to 2 nM and volumes were mixed together based on number of libraries in the pool to make the sequencing sample pool. A PhiX control library was spiked in at 0.2% before sequencing.
?. [Library Preparation]
Homebrew Library PreparationIllumina sequencing libraries were prepared using a homebrew library preprartion protocol modified from previously reported tagmentation-based protocols2,3 and a PIPETMAN® M Connected electronic pipette.Pipetting plan:  Tagmentation was carried out by mixing each well with 1 uL of 1.6x Homebrew Tn5 Tagmentation Buffer and 0.2 uL of Tn5 enzyme at (UC Berkeley Macrolab), then incubated at 55 °C for 3 min. The reaction was stopped by adding 0.4 μl 0.1% sodium dodecyle sulfate(Fisher Scientific, BP166-500) and centrifuging at room temperature at 3,220g for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of KAPA HiFi Non-Hot Start Master Mix (Kapa Biosystems). PCR amplification was carried out on a ProFlex 2x384 thermal cycler (Thermo Fisher) using the following program: 1) 72 °C for 3min2) 95 °C for 30 s3) 12 cycles :    98°C for 10s    67°C for 30s    72°C for 1min4)72°C for 5min. 1.6x Homebrew Tn5 Tagmentation Buffer16 mM Tris-HCl, pH 7.4 (Lonza, 51247)16 mM MgCl2 (Invitrogen, AM9530G)8% v/v N,N-dimethyformamide (Sigma-Aldrich, 227056-1L)KAPA HiFi Non-Hot Start Master Mix0.067 U/μL KAPA HiFi Standard Polymerase (Kapa Biosystems, KK2102)3.33x KAPA HiFi Fidelity Buffer(Kapa Biosystems, KK2102)1 μM dNTP Mix (Kapa Biosystems, KK2102)2. Hennig, B. P. et al. Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3 Genes Genomes Genetics 8, 79–89 (2018). 3. Picelli, S. et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res 24, 2033–2040 (2014).
?. [RNA Plating and Normalization]
Extracted bulk RNA samples were plated in 96 well hard-shell PCR plates (Bio-Rad HSP9601) by PIPETMAN® M Connected electronic pipette and normalized to 25 ng/μL in RNAse free water and Recombinant RNase Inhibitor. Two μL aliquots of normalized RNA was transferred to a new 96-well plate and mixed with 2 μL of  RNA Buffer Master Mix. These sample plates were incubated at 72 ˚C for 3 mins and slowly cooled to facilitate oligo-dT annealing. (Optional: Plates were stored at -80˚C until processing.)Pipetting plan: RNA Buffer Master Mix 2 U/μL Recombinant RNase Inhibitor (Takara Bio, 2313B)5 mM dNTP mix (Thermo Fisher, R0193)5 μM Oligo-dT30VN (Integrated DNA Technologies, 5′AAGCAGTGGTATCAACGCAGAGTACT30VN-3′)
?. [cDNA Synthesis]
cDNA Cleaning, Reformatting,  and QuantificationThe amplified products were purified once with 0.7x AMPure beads (Fisher, A63881) by hand and reformatted from 96-well plates to new 384-well hard-shell PCR plates (Bio- Rad HSP3901) by hand for quanitification and further processing. Concentrations were measured with a dye-fluorescence assay (Quant-iT dsDNA High Sensitivity kit; Thermo Fisher, Q33120) on a SpectraMax i3x microplate reader (Molecular Devices). Samples were normalized by two rounds of dilutions to 1.5 ng/μl and then 0.5 ng/μl in 384-well Echo 384-well Polypropylene Microtiter plates (Labcyte, PP-0200). For every sample,  0.8 μl of normalized cDNA was transfered to a new 384-well plate using a Viaflow 384  Multichannel Pipette (Integra).
?. [Library Pooling, QC, and Sequencing]
To ensure relatively even sequencing depth across all libraries, an initial sequencing run was performed on the iSeq 100 Sequencing System (Illumina) using 2 x 100-bp paired end reads and 2 x 12-bp index reads with iSeq 100 i1 300-cycle single kit (Illumina, 20021533). The pooled volume of individual bulk libraries was adjusted according to the sequencing depth observed from this initial run, and libraries were subsequently purified and QC-ed as described in Step 7.
?. [Data Processing]
Sequences from the NovaSeq were de-multiplexed using bcl2fastq version 2.20. Reads were aligned to the GRCm39.p6 (mus musculus) genome with Gencode v.M19 annotations using using STAR version 2.5.2b with parameters TK. Gene counts were produced using HTSEQ version 0.6.1p1 with default parameters, except “stranded” was set to “false”, and “mode” was set to “intersection-nonempty”.